Isoflurane enhances autophagy by activating AMPK/ULK1, inhibits NLRP3, and reduces cognitive impairment after cerebral ischemia-reperfusion injury in rats

doi:10.21203/rs.3.rs-1988668/v1

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Research Article

Isoflurane enhances autophagy by activating AMPK/ULK1, inhibits NLRP3, and reduces cognitive impairment after cerebral ischemia-reperfusion injury in rats

https://doi.org/10.21203/rs.3.rs-1988668/v1

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Background

Cerebral ischemia-reperfusion injury (CIRI) is an important factor affecting the prognosis of patients with ischemic stroke. This study aimed to observe whether isoflurane posttreatment enhances autophagy after focal CIRI in rats by activating the AMPK/ULK1 signaling pathway, thereby inhibiting NLRP3 inflammation. The body releases inflammatory factors to reduce inflammation, thereby reducing neurological damage and improving cognitive and memory functions.

Methods

Eighty male SD rats were randomly divided into 5 groups: sham operation group (Sham), model group (MCAO), isoflurane posttreatment + model group (M + ISO), autophagy inhibitor Baf-A1 + isoflurane posttreatment + model group (M + I + B), AMPK inhibitor Compound C + isoflurane posttreatment + model group (M + I + C).

Results

Compared with the sham group, the MCAO group exhibited decreased neurobehavioral scores and cognitive memory function (P < 0.05). Compared with the MCAO group, the neurobehavioral score of rats in the M + ISO group was significantly reduced, the expression of AMPK, ULK1, Beclin1, and LC3B protein was significantly increased, and cognitive and memory function was significantly improved (P < 0.05). Compared with the M + ISO group, the neurobehavioral scores and the protein expression of NLRP3, IL-1β and IL-18 in the M + I + B and M + I + C groups increased significantly (P < 0.05).

Conclusions

This research indicated that isoflurane posttreatment may enhance autophagy by activating the AMPK/ULK1 signaling pathway and further inhibit the release of inflammatory factors from NLRP3 inflammasomes, thereby improving neurological function and cognitive impairment after focal CIRI in rats and exerting a protective effect on the brain.

Autophagy

AMPK

ULK1

NLRP3

Isoflurane

Cerebral ischemia-reperfusion injury

Cerebral ischemic stroke (CIS) has the characteristics of high morbidity, high disability and high mortality and has become the second leading cause of death in the world (Wu, et al., 2021). Despite the basic research and clinical diagnosis and treatment carried out, CIS has been ongoing for many years, but CIS is still one of the most important public health problems(Lyden, et al., 2021). Opening blood vessels in time and restoring blood perfusion are the main treatment principles of CIS (Yin, et al., 2021). However, when ischemic brain tissue restores blood perfusion, ischemic damage is further aggravated. This phenomenon is called cerebral ischemia-reperfusion injury (CIRI) and can often lead to cell inflammation, necrosis, and apoptosis (Nasoohi, et al., 2019), and eventually lead to neurological damage such as cognition, memory, learning and so on.

During CIRI, stress can activate autophagy, and neurons are particularly sensitive to changes in autophagy activity. Studies have shown that the activation and upregulation of autophagy reverses neurological deficits (Li, et al., 2018) and plays an important role in neuroprotection (Wen, et al., 2019). Autophagy is an important catabolic program that processes various macromolecular cellular contents to maintain homeostasis, while AMP-activated protein kinase (AMPK) is an important metabolic regulator that simultaneously inhibits anabolism pathway and activate catabolic pathways to achieve energy homeostasis. AMPK recruitment to lysosomes may be critical for the integration of nutrient signaling and autophagy. A large number of studies have shown that autophagy is the downstream target of AMPK, and it is believed that the activation of autophagy mediated by AMPK is a potential protective mechanism against brain injury (Inoki, et al., 2012, Jiang, et al., 2014, Jiang, et al., 2015). In addition, unc-51-like kinase 1 (ULK1) is considered to be an important autophagy initiator (Lee, et al., 2010). In mammals, the ULK1 complex can initiate phagosome membrane structure and recruit subsequent ATG proteins to form autophagosomes. Therefore, when cell energy is lacking, AMPK can directly activate the ULK1 complex to induce autophagy (Hurley and Young, et al., 2017). In addition, the NLRP3 inflammasome also plays a crucial role in brain injury induced by ischemia-reperfusion (Palomino-Antolin, et al., 2021). Some studies have also reported that the NLRP3 inflammasome-mediated inflammatory response may be regulated by autophagy (Lei, et al., 2021, Yu, et al., 2021). Therefore, exploring the activation of autophagy after nerve injury and its influence on the further regulation of inflammation will guide us in the treatment of CIRI.

Preliminary research by this research group has confirmed that inhaled anesthetic isoflurane (ISO), which is widely used in clinical anesthesia, plays an important protective role in central nervous system damage. It may be related to multiple signaling pathways and oxidative stress, and cell inflammation is closely related to apoptosis (Peng, et al., 2019, Yin, et al., 2020, Zhang, et al., 2019). Through the research group's previous research, we have found that ISO can improve CIRI by reducing cell edema, promoting angiogenesis, reducing cell inflammation, apoptosis and pyrolysis, etc., and has a protective effect on the brain (Ge, et al., 2021, Peng, et al., 2019, Yang, et al., 2020, Yuan, et al., 2018, Zhang, et al., 2021). However, in CIRI, whether isoflurane can activate the AMPK/ULK1 signaling pathway and the autophagy mediated by it can exert a protective effect on the brain are not yet clear. This study intends to establish an MCAO model to explore the role of autophagy and the AMPK/ULK1 signaling pathway in isoflurane-induced neuroprotection and the relationship between autophagy and the NLRP3 inflammasome in neuroprotection (Fig. 1).

Ethics Statement

All experimental animal protocols followed the regulations of the People's Republic of China on the Administration of Laboratory Animals. The experimental procedures were approved by the Animal Research Committee by Shihezi University (protocol A2019-025-01).

Animals and Grouping

Healthy and clean male Sprague-Dawley (SD) rats weighing 200-250g were chosen, all provided by the Experimental Animal Center of Shihezi University School of Medicine. The breeding room temperature is between 21–24°C, the relative humidity is 40%-70%, and the supply of water and food is sufficient. 4 rats were housed in each cage, and the cages were cleaned once a week. A total of 87 rats were selected, after excluding the dead rats, the remaining 80 rats were randomly divided into 5 groups with 16 rats in each group: Sham operation group (Sham), model group (MCAO), isoflurane posttreatment + model group (M + ISO), autophagy inhibitor Baf-A1 + isoflurane posttreatment + model Group (M + I + B), AMPK inhibitor Compound C + Isoflurane posttreatment + model group (M + I + C). All experimental rats were treated with 3% pentobarbital sodium by intraperitoneal injection of 5 mg/100 g for basic anesthesia.

Middle Cerebral Artery Occlusion (MCAO) Model

The anesthetized rat was fixed in the supine position on a 37°C constant temperature operating table, and the MCAO model was established by referring to the modified Longa method (Tian and Mao, et al., 2022). After disinfection of the neck skin, a median incision was made, and the right common carotid artery (CCA), external carotid artery (ECA) and internal carotid artery (ICA) were separated and exposed. Cut a small oblique incision near the bifurcation of the internal and external carotid arteries of CCA, insert a 3 − 0 nylon thread plug, and insert the depth of 18.0 ± 1.0 mm from the bifurcation. In the Sham group, only the CCA, ECA, and ICA on the right side were separated, the nylon thread bolt was not inserted. The other operations were the same as those in the model group. After 90 minutes of embolization, the nylon thread plug was carefully pulled out to create a reperfusion model, and the reperfusion time was 24 hours. The isoflurane posttreatment group (M + ISO, M + I + B, M + I + C) was post-treated with 1.5% isoflurane for 60 minutes immediately after reperfusion. The inhibitor group (M + I + B, M + I + C) was injected with Baf-A1 (10 µl/piece, 0.1ug/piece, S1413, Selleck) and Compound C (10 µl/piece, 20ug/piece, S7306, Selleck) 30 minutes before the model was established. The modeled rats were placed on the thermal insulation and resuscitation blanket. When awakened, they were returned to the cage and reared alone, with free eating and drinking.

Neurobehavioral experiment

The modified Neurological Severity Scores (mNSS) standard was used to evaluate the neurological function of rats 24 hours after ischemia-reperfusion through four aspects: exercise test, sensory test, balance beam test, loss of reflex and abnormal movement. The total score is 18 points, the higher the score, the more severe the nerve function damage.

Analyze the learning, cognitive and memory functions of rats by avoiding the dark experiment. Adaptation: The rat was put into the bright room, opened the door hole between the bright and dark rooms, let it move freely for 3 ~ 5 minutes, then put it back into the cage. Training: The next day, put the rat into the bright room. Since rats have the characteristics of darkening and evil light, they will quickly enter the dark room. After the rat entered the dark room, closed the door and started electrical stimulation at the same time. The incubation period of the rat entered the dark room was recorded from the bright room, which can be used as an indicator to judge whether there was a difference in the behavior of each rat. Testing: 24 hours after training, the rats were placed in the light chamber again. The time from the time the rat was put into the bright room to the first time it entered the dark room voluntarily was recorded as the step through latency (STL), and the maximum latency was set to 300s. And the number of times the rat entered the darkroom within 300 seconds was recorded, which was called error Times (ET). If the rat did not step into the dark room within 300s, the test was terminated and the next one was continued. Normal rats will not enter the dark room voluntarily due to the existence of electrical stimulation memory, while rats with neurological impairment have reduced learning and memory ability, and will enter the dark room in a short period of time and repeatedly enter and exit. Therefore, if the cognitive learning and memory function is impaired, the STL is significantly smaller than that of the control group, and the ET is significantly larger than that of the control group.

Laser speckle imaging (LSI)

Changes in cerebral blood flow (CBF) in rats were observed by a laser speckle imaging system. Place the anesthetized rat in the prone position, cut the scalp along the center, separate and expose the surface of the skull, and use the laser speckle imaging system (Moore FLPI-2, Beijing, China), using a laser diode (785 nm; Moore FLPI − 2, Beijing, China) obtained images at 23fps (exposure time t = 5ms). Changes of CBF in rats were observed, sutures and resuscitates after completion.

2,3,5-triphenyltetrazolium chloride (TTC) Staining

TTC staining measures the volume of cerebral infarction. The anesthetized rats were decapitated and put to death. The brains were quickly removed and cut into five brain slices approximately 2 mm thick rostro-caudal, and quickly placed in 2% TTC solution (Servicebio, Wuhan, China) at a constant temperature of 37°C to avoid incubate in light for 30 min, and fixed in 10% paraformaldehyde (Servicebio, Wuhan, China) after staining. Stored in a 4° refrigerator for 24 hours. After 24 hours, take it out and take a photo. The infarct area of the brain slice was measured with the medical image analysis software Image J (Stuttgart, Germany) (red indicates normal brain tissue, and pale indicates the infarct area). The infarct volume (%) was calculated as the infarct area relative to the contralateral hemisphere area in each slice.

Western Blotting (WB) Analysis

The anesthetized rats were sacrificed by decapitation and the brain was quickly taken out, placed in 4° artificial cerebrospinal fluid to rapidly strip the hippocampus tissue, put into a cryopreservation tube, marked and stored in a -80° refrigerator for later use. The frozen hippocampal tissue was taken out and weighed and placed in a clean EP tube, RIPA lysis solution and protease inhibitor PMSF were added, and the hippocampal tissue was crushed using an ultrasonic oscillator, and the whole process was operated on ice to ensure protein activity. The extracted supernatant was placed in a pre-cooled 4° centrifuge, centrifuged at 12,000 rpm for 5 min, and the centrifuged supernatant was quantified by BCA method to prepare a sample with a protein content of 3 mg/mL (Chen, et al., 2021), and loading buffer was added to the sample. Then put it into boiling water and cook for 10min. After cooling, the electrophoresis samples were divided and stored in a -80° refrigerator for later use. Take samples for electrophoresis, and the quantity of protein is 15ug. After electrophoresis, the protein was transferred to PVDF membrane by wet transfer method, and blocked with 5% skim milk for 2 hours, and then add rabbit anti-LC3B (1:2000, Abcam, ab192890), rabbit anti-Beclin1 (1:1000, Abcam, ab62557), rabbit anti-AMPK (1:1000, Abcam, ab32047), rabbit anti-ULK1 (1:1000, Abcam, ab240916), rabbit Anti-NLRP3 (1:1000, Abcam, ab263899), rabbit anti-IL-1β (1:1000, Abcam, ab216995), rabbit anti-IL-18 (1:1000, Abcam, ab207323) and rabbit anti-GAPDH (1:1000, Bioss, 0755R, Beijing, China), incubate overnight at 4°C in a shaker. After washing with TBST buffer the next day, the PVDF membrane were hybridized with horseradish peroxidase-labeled secondary antibody (goat anti-rabbit, 1:1500, Bioss, bs-0295G-SA, Beijing, China) for 2 hours. Pictures were taken using a chemiluminescence imaging system (Tanon5200, Shanghai, China). Image J (Stuttgart, Germany) image analysis software was used to calculate the ratio of the gray value of the target protein to the internal reference protein for statistical analysis.

Immunofluorescence (IF) staining

The anesthetized rats were fixed in the supine position on the operating table, and the brains were harvested by cardiac perfusion. The brain tissues were fixed in 10% paraformaldehyde (Servicebio, Wuhan, China) and stored in a 4° refrigerator. After 3–5 days, the brain tissue was dehydrated in 20% sucrose formalin solution for 2 days, stored at 4°, embedded in paraffin after removal, and sliced with a paraffin microtome. The paraffin sections were dewaxed in xylene and alcohol of different concentrations in turn, then placed in citric acid repair solution (Servicebio, Wuhan, China) for high temperature repair, and blocked with 20% BSA for 1 hour. Rabbit anti-LC3B (1:200, Abcam, ab192890), rabbit anti-Beclin1 (1:100, Abcam, ab62557), rabbit anti-AMPK (1:250, Abcam, ab32047), and rabbit anti-ULK1 (1:100, Abcam, ab240916) were added, and the sections were incubated overnight at 4°C. The next day, fluorescence-labeled secondary antibody (goat anti-rabbit 488, 1:1000, Abcam, ab150077) was incubated at 40°C for 1 hour, stained with the nuclear fluorescent stain propidium iodide (PI, Servicebio, Wuhan, China) for 5 minutes, and then anti-fluorescence quenching mounting tablets (Servicebio, Wuhan, China) were added. Finally, a laser scanning confocal microscope (PerkinElmer, USA) was used to observe the expression of the target protein in the CA1 area of the rat hippocampus, to compare the fluorescence intensity and to perform statistical analysis.

Transmission electron microscopy (TEM)

Rat hippocampal tissue was collected after reperfusion for 24 hours, the sample size was approximately 2 mm × 2 mm, and the tissue was placed in 2.5% glutaraldehyde special fixative for electron microscopy (P1126, Solarbio, Beijing, China). After staining and dehydration, Epon resin (Brand, Germany) was added and incubated for 24 hours. After preparing slices with a thickness of about 50–70 nm using an ultramicrotome (RMC-PXL, USA), the autophagosome morphology was observed with a transmission electron microscope (FEI Tecnai G2 F30).

Statistics Analysis

GraphPad Prism 8.0.2 software (San Diego, CA, USA) was used for statistical analysis. Normality test was applied before one-way Analysis of Variance (ANOVA) for multiple groups comparison. Data fitting a parametric distribution were tested for significance using analysis of Student’s t-test and one-way analysis of variance (ANOVA). Nonnormally distributed measurement data were tested by nonparametric tests. P < 0.05 indicates that the difference is statistically significant.

MCAO model was successfully prepared

Through laser speckle observation of cerebral blood flow and TTC measurement of cerebral infarction area, we can see that the bilateral cerebral blood flow of rats in the Sham group was normal, while the cerebral blood flow perfusion on the injured side of the MCAO group was significantly insufficient (Fig. 2, P < 0.0001). In addition, compared with the sham group, the mNSS behavior score of the MCAO group after 24 hours was significantly increased, indicating that the rats' nerve function was damaged (Fig. 3B-F, P < 0.0001). The above results indicate that the MCAO model was successfully made in this experiment, and the damage effect was stable.

Isoflurane posttreatment can upregulate the autophagy activity of neuronal cells after CIRI in rats

First, through Western blotting, it can be seen that the expression of autophagy-related proteins Beclin1 and LC3B in the MCAO group was significantly higher than that in the Sham group, and immunofluorescence also has consistent results, indicating that CIRI can activate the autophagy activity of rat hippocampal neuronal cells (Fig. 4, Western blotting: Beclin1 P = 0.0012, LC3B P = 0.0023; Immunofluorescence: Beclin1 P < 0.0001, LC3B P = 0.0016). Next, in the M + ISO group, the expression of Beclin1 and LC3B further increased, and when autophagy was inhibited, in the M + I + B group, the expression levels of Beclin1 and LC3B were significantly lower than that of the M + ISO group, indicating that isoflurane posttreatment can further promote neuronal autophagy after injury (Fig. 4, Western blotting: Beclin1 P = 0.0015, LC3B P = 0.0393; Immunofluorescence: Beclin1 P = 0.0005, LC3B P < 0.0001). In addition, the observation of cell morphology by transmission electron microscopy also showed that the morphology of rat hippocampal neurons before injury was normal, and a large number of synapses were visible (Fig. 5A-C). Autophagolysosomes appeared after injury, and the damaged cytoplasm of neurons presents autophagy-like vacuoles, multiple vacuoles appear in the cytoplasm, mitochondrial morphology is abnormal, and cell synapses are also significantly reduced (Fig. 5D-F). The area of autophagolysosomes were also significantly increased in isoflurane-treated rat hippocampal tissue (Fig. 5G-I). At the same time, the performance of rat neurological function also has obvious changes in behavioral experiments: it can be seen from the scores of motor test, sensory test, balance beam test, loss of reflex and abnormal movement in the mNSS score. Compared with the MCAO group, the mNSS score of the M + ISO group was significantly reduced, compared with the M + I group, the score of the M + I + B group was visibly increased (Fig. 3C-F, Western blotting: Beclin1 P = 0.0002, LC3B P = 0.0007; Immunofluorescence: Beclin1 P < 0.0001, LC3B P < 0.0001). At the same time, the dark avoidance experiment also had consistent experimental results: the stepping latency (STL) of rats in the M + ISO group was significantly longer than that of the MCAO group, and the number of errors entering the dark box (ET) was also evidently reduced, while in the M + I + B group, STL was distinctly shortened, and ET was obviously increased (Fig. 3K-L, all P < 0.0001), indicating that the cognitive and memory function of rats was evidently improved by isoflurane posttreatment. As the autophagy activity was inhibited, the cognitive and memory function of rats after isoflurane treatment also decreased significantly. These results showed that isoflurane can reduce nerve damage after cerebral ischemia-reperfusion by upregulating autophagy and improve the cognitive and memory function of rats.

Isoflurane posttreatment upregulates autophagy after CIRI in rats by activating the AMPK/ULK1 signaling pathway

Through Western blotting and immunofluorescence experiments, it can be seen that the expression of signal pathway related protein AMPK and its downstream molecules ULK1, and the expression of autophagy-related proteins Beclin1 and LC3B in the MCAO group are significant higher than those in the Sham group, indicating that cerebral ischemia-reperfusion injury may further upregulate the autophagy of rat hippocampal neuronal cells by activating AMPK/ULK1 signaling pathway (Fig. 6, Western blotting: AMPK P = 0.0006, ULK1 P = 0.0014, Beclin1 P = 0.0039, LC3B P = 0.0019; Immunofluorescence: AMPK P < 0.0001, ULK1 P < 0.0001, Beclin1 P = 0.0003, LC3B P = 0.0005). After isoflurane treatment, the expression of AMPK, ULK1, Beclin1 and LC3B in the M + ISO group was significantly higher than those of MCAO group (Fig. 6, Western blotting: AMPK P = 0.0086, ULK1 P = 0.0082, Beclin1 P = 0.0055, LC3B P = 0.0035; Immunofluorescence: AMPK P < 0.0001, ULK1 P = 0.0227, Beclin1 P = 0.001, LC3B P < 0.0001). Compared with M + ISO group, after AMPK inhibitor was used in M + I + C group, the expression of AMPK and ULK1 decreased significantly. The expression of the autophagy-related proteins Beclin1 and LC3B in the hippocampus of rats was also significantly reduced (Fig. 6, Western blotting: AMPK P < 0.0001, ULK1 P = 0.0001, Beclin1 P = 0.0002, LC3B P < 0.0001; Immunofluorescence: AMPK P < 0.0001, ULK1 P < 0.0001, Beclin1 P < 0.0001, LC3B P < 0.0001), and behavioral damage was obvious (Fig. 3B, K, L, mNSS: P < 0.0001, STL: P < 0.0001, ET: P < 0.0001). The above results indicate that isoflurane posttreatment may further upregulate neuronal autophagy after CIRI by activating the AMPK/ULK1 signaling pathway, thereby reducing neuronal damage in the hippocampus of rats and improving cognitive and memory function after injury.

After CIRI in rats, isoflurane posttreatment upregulates autophagy and inhibits the release of inflammatory factors from NLRP3 inflammasome

The results of Western blotting experiment showed that, compared with the MCAO group, the expression of NLRP3 inflammasome and related inflammatory factors IL-1β and IL-18 in the M + ISO group was significantly decreased, indicating that isoflurane posttreatment can reduce the inflammatory injury caused by cerebral ischemia reperfusion (Fig. 7, NLRP3 P = 0.0014, IL-1β P = 0.0135, IL-18 P = 0.0005). Moreover, compared with the M + ISO group, the expression of NLRP3, IL-1β and IL-18 in the M + I + B group was visibly increased, indicating that after autophagy is inhibited, inflammation further increases (Fig. 7, NLRP3 P = 0.0018, IL-1β P = 0.0079, IL-18 P = 0.00435), that is to say, the alleviating effect of isoflurane posttreatment on inflammation may be achieved through the upregulation of autophagy.

Isoflurane posttreatment upregulates autophagy after focal CIRI in rats through the AMPK/ULK1 signaling pathway, thereby inhibiting the release of inflammatory factors by the NLRP3 inflammasome

Through the results of Western blotting experiment, it can be seen that the expressions of NLRP3, IL-1β and IL-18 in the M + I + C group given AMPK pathway inhibitors were significantly increased compared with the M + ISO group, indicating that the inhibitory effect of isoflurane posttreatment on NLRP3 inflammasome may be achieved by activating AMPK/ULK1 signaling pathway and upregulating autophagy after CIRI in rats (Fig. 8, NLRP3 P = 0.0008, IL-1β P = 0.0020, IL-18 P = 0.0043).

Since Cerebral ischemia-reperfusion injury (CIRI) can cause severe brain tissue damage, it is an important factor leading to poor prognosis such as disability or death in patients with ischemic stroke (Wang and Wang, et al., 2021), therefore, it is particularly important to explore treatments that can effectively promote the recovery of patients. Isoflurane is a safe and reliable inhalation anesthetic gas. In recent years, due to its brain protective effect, it has been widely studied. Our research group also found in a preliminary study that 1.5% isoflurane posttreatment showed better brain protection (Wang, et al., 2016). In this study, we verified the success of the MCAO model through laser speckling and TTC, and behavioral experiments also showed that the damaging effect of the reperfusion model was relatively stable. Among them, in behavioral experiments we found that after 24 hours of cerebral ischemia-reperfusion in rats, the total mNSS score increased significantly. In the exercise test, the rats turned around or dumped to the paraplegic side when walking, and could not hold the balance beam tightly in the balance beam test, and easy to fall, in addition, the auricle reflex and corneal reflex were significantly weakened, and showed a clear panic response to noise. In the dark avoidance experiment, although the rats in the MCAO group had undergone adaptation and dark room electrical stimulation training, they still entered the dark room in a short period of time, and repeatedly entered within 300s, it indicated that the cognitive, learning and memory abilities of rats were significantly decreased after CIRI. In CIRI, hippocampal tissue is proven to be more sensitive to ischemia and hypoxia (Zhang, et al., 2019), therefore, 1.5% isoflurane posttreatment was used to discuss the changes and regulatory mechanisms of neuronal autophagy activity in the hippocampal CA1 region of CIRI rats.

Autophagy is an important cell catabolism program that can play a protective role in a variety of cells, degrade waste proteins and damaged organelles, regulate cell growth, metabolism and survival, and maintain cell homeostasis (Boya, et al., 2013, Ha, et al., 2017). Studies have shown that CIRI can activate the autophagy of nerve cells (Ping, et al., 2021), and the further upregulation of autophagy can significantly improve nerve function after injury and play an important role in neuroprotection (Li, et al., 2018, Wen, et al., 2019). Microtubule-associated protein light chain 3 (LC3), the mammalian homolog of yeast ATG8, is synthesized and cleaved into LC3-I, which activates transcription factor 4 protease, thereby activating the autophagy process. LC3-I is linked to the lipid phosphatidylethanolamine to form LC3-II, and the protein level of its membrane component is often used to measure autophagic activity. The LC3 family includes three members, LC3A, LC3B, and LC3C, of which LC3B is a well-known marker of autophagy and one of the most widely studied members. Beclin1 is an important autophagy-related molecule in I/R injury, and plays an important role in inducing the initiation of autophagy and promoting autophagosome maturation. Therefore, in this study, LC3B and Beclin1 were selected as the main protein molecules to observe the changes of autophagy activity. During the study, we found that the expression of the level of autophagy-related proteins Beclin1and LC3B in MCAO group rats were significantly increased as demonstrated by Western blotting and immunofluorescence experiments, which further supports the conclusion that CIRI can activate the autophagy activity of neural cells in injured rats. In addition, when using isoflurane posttreatment, we found that the expression of Beclin1 and LC3B further increased. At the same time, mNSS scores and dark avoidance experiment showed that the neurobehavior and learning and memory function of rats were obviously improved, which indicates that isoflurane treatment can improve the neurological damage of rats by further upregulating the autophagy activity of rat hippocampal neurons after CIRI.

AMP-activated protein kinase (AMPK) is an evolutionarily conserved serine/threonine protein kinase that can inhibit cellular energy expenditure pathways and simultaneously activate compensatory energy production procedures to achieve energy homeostasis (Sheng, et al., 2014). Studies have shown that AMPK-mediated autophagy activation is a potential protective mechanism in the early stage of brain injury (Inoki, et al., 2012, Jiang, et al., 2014, Jiang, et al., 2015). In the absence of energy, AMPK can directly activate the ULK1 complex to induce autophagy (Hurley and Young, et al., 2017). In this study, we found that when isoflurane was used, the expression of AMPK and ULK1 was significantly increased compared with that in the model group, and the expression of the autophagy-related proteins Beclin1 and LC3B was also evidently increased. In addition, behavioral experiments also showed that the neurological damage in rats was significantly improved. However, when AMPK was inhibited, the expressions of Beclin1 and LC3B were significantly decreased, and behavioral experiments also showed that the neurological function of rats was severely damaged, indicating that isoflurane posttreatment may upregulate autophagy of neuronal cells after CIRI by activating the AMPK/ULK1 signaling pathway, thereby exerting brain protection.

In addition, studies have shown that the NLRP3 inflammasome plays a key role in the development of CIRI, it can recruit and activate procaspase-1 to generate active caspase-1, and then convert cytokine precursors pro-IL-1β and pro-IL-18 into mature and biologically active IL-1β and IL-18, respectively, which Once activated, it triggers a series of inflammatory responses (Palomino-Antolin, et al., 2021), thus targeting control of the activation of NLRP3 inflammation is of great importance in the treatment of CIRI. Some studies have also reported that the activation and upregulation of autophagy can well inhibit the inflammatory response mediated by NLRP3 inflammasome (Li, et al., 2021, Yu, et al., 2021). In this study, we found that when CIRI rats were post-treated with isoflurane, the expression of the NLRP3 inflammasome and its mediated IL-1β and IL-18 were significantly reduced. When specific autophagy inhibitors Baf-A1 and the AMPK inhibitor Compound C were used, the expression of NLRP3, IL-1β and IL-18 was obviously increased, indicating that isoflurane posttreatment can inhibit the release of inflammatory factors from NLRP3 inflammasome, and this inhibitory effect may be achieved by isoflurane through the up-regulation of autophagy by the AMPK/ULK1 signaling pathway. At the same time, behavioral experimental results also show that isoflurane improves the neurobehavior and cognitive memory learning function of CIRI rats by regulating autophagy and inhibiting NLRP3-mediated inflammation.

Studies have shown that overexpression or downregulation of autophagy can cause damage to cells and even cell death. In this study, only autophagy inhibitors and AMPK inhibitors were used. If the group of agonists can be increased, the verification of the research results can be more perfect. In addition, if combined in vitro and in vivo experiments can be performed at the same time, the study conclusions can be better verified, which is also a deficiency of this study.

Our results suggested that autophagy in hippocampal neurons is activated after CIRI in rats. Isoflurane posttreatment may further upregulate autophagy by activating the AMPK/ULK1 signaling pathway, and the upregulated autophagy can further inhibit NLRP3 inflammation releases IL-1β, IL-18 and other inflammatory factors, thereby reducing the damage of hippocampal neurons in CIRI rats and improving the rats' neurobehavioral function and cognitive learning function. In general, the results of this study may provide effective evidence for the treatment of CIRI-related diseases in terms of targeted regulation of autophagy and inhibition of inflammation.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

The present study was approved by the Animal Experimental Committee of the First Affiliated Hospital, School of Medicine, Shihezi University.

CONSENT FOR PUBLICATION

Not applicable.

AVAILABILITY OF DATA AND MATERIALS

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

COMPETING INTERESTS

The authors declare that they have no competing interests in this work.

FUNDING

This study was supported by the National Natural Science Foundation of China (82160235 and 81860249).

AUTHOR CONTRIBUTIONS

SW, J-W Y, and J-W Z contributed to the study design and revised the manuscript. J-W Z, NL, and XZ conducted the experiment and collected and analyzed the data. K-T M, YL and X-L Q provided assistance in experiment performing. J-W Z and R-X W wrote the manuscript. All authors read and approved the final manuscript.

ACKNOWLEDGMENTS

First of all, a very sincere thanks to SW and J-W Y for their contributions to the study design and data analysis. Secondly, thanks to NL, XZ and L-X Q for their efforts in the implementation of the study. Finally, thanks to K-T M, YL and X-R W for their guidance on experimental methods.

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Isoflurane enhances autophagy by activating AMPK/ULK1, inhibits NLRP3, and reduces cognitive impairment after cerebral ischemia-reperfusion injury in rats

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Middle Cerebral Artery Occlusion (MCAO) Model

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