H2S Attenuates Sleep Deprivation‑Induced Cognitive Impairment by Reducing Excessive Autophagy via Hippocampal Sirt‑1 in WISTAR RATS

Shan Gao1 · Yi‑Yun Tang1 · Li Jiang1,2 · Fang Lan2,3 · Xiang Li4 · Ping Zhang1,2 · Wei Zou1,2 · Yong‑Jun Chen1,2 · Xiao‑Qing Tang1,3

Received: 24 November 2020 / Revised: 5 March 2021 / Accepted: 24 March 2021
© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2021

Abstract
Sleep deprivation (SD) is widespread in society causing serious damage to cognitive function. Hydrogen sulfide (H2S), the third gas signal molecule, plays important regulatory role in learning and memory functions. Inhibition of excessive autophagy and upregulation of silent information regulator 1 (Sirt-1) have been reported to prevent cognitive dysfunction. Therefore, this present work was to address whether H2S attenuates the cognitive impairment induced by SD in Wistar rats and whether the underlying mechanisms involve in inhibition of excessive autophagy and upregulation of Sirt-1. After treat- ment with SD for 72 h, the cognitive function of Wistar rats was evaluated by Y-maze, new object recognition, object loca- tion, and Morris water maze tests. The results shown that SD-caused cognitive impairment was reversed by treatment with NaHS (a donor of H2S). NaHS also prevented SD-induced hippocampal excessive autophagy, as evidenced by the decrease in autophagosomes, the down-regulation of Beclin1, and the up-regulation of p62 in the hippocampus of SD-exposed Wistar rats. Furthermore, Sirtinol, an inhibitor of Sirt-1, reversed the inhibitory roles of NaHS in SD-induced cognitive impairment and excessive hippocampal autophagy in Wistar rats. Taken together, our results suggested that H2S improves the cognitive function of SD-exposed rats by inhibiting excessive hippocampal autophagy in a hippocampal Sirt-1-dependent way.
Keywords Sleep deprivation · Cognitive impairment · Hydrogen sulfide · Autophagy · Silent information regulator 1

Shan Gao, Yi-Yun Tang, and Li Jiang are contributed equally to this work.
 Ping Zhang
[email protected]; [email protected]
 Xiao-Qing Tang
[email protected]; [email protected]
1 Hengyang Key Laboratory of Neurodegeneration
and Cognitive Impairment, Institute of Neuroscience, Hengyang Medical College, University of South China, 28 W Changsheng Road, Hengyang 421001, Hunan, P. R. China
2 Department of Neurology, Affiliated Nanhua Hospital, University of South China, 336 E Dongfeng Road, Hengyang 421001, Hunan, P. R. China
3 Institute of Neurology, the First Affiliated Hospital, University of South China, Hengyang 421001, Hunan,
P. R. China
4 Department of Anesthesiology, the First Affiliated Hospital, University of South China, Hengyang 421001, Hunan,
P. R. China

Introduction
Sleep plays a key role in maintaining human health, espe- cially in gene expression, protein synthesis and memory consolidation [1]. Sleep deprivation (SD), meaning insuffi- cient and irregular sleep, is an increasing trend and had been thought as a life-threatening factor [2, 3]. Increasing studies report that SD interferes with almost all cognitive processes in humans [4], such as impairing Long-term potentiation (LTP) of hippocampus [4, 5]. SD leads to glutamatergic signaling and cholinergic signaling reduction, cAMP signal- ing and CREB-mediated gene transcription inhibition, which provide theoretical bases for SD-led impairment in learning and memory [5]. Therefore, further research for the potential therapeutic method to prevent SD-caused cognitive dysfunc- tion is urgently needed.
Hydrogen sulfide (H2S), recognized as the third gas signal molecule following NO and CO, is thought as a novel neu- romodulator [6–8]. In mammalian, cystathionine-β-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (3-MST)

contribute the synthesize of H2S in brain and mitochondria [9]. H2S promotes the development of hippocampal long- term potentiation (LTP) [10] and long-term depression (LTD) [11], improves synaptic plasticity [12], and regu- lates learning, memory and cognitive function [13]. These findings indicate that H2S has the potential role to antago- nize cognitive dysfunction. Studies have shown that H2S has therapeutic benefits in cognitive impairment induced by subarachnoid hemorrhage [14], Alzheimer’s disease
[15] and vascular dementia [16]. Our previous studies also showed that H2S ameliorates cognitive dysfunction induced by diabetes [17], acute liver failure [18] and formaldehyde- exposure [19]. Overall, H2S is an important target to pre- vent and treat cognitive dysfunction. At present, it is not clear whether H2S improves SD-induced cognitive impair- ment. The purpose of this article is to explore whether H2S resists SD-induced cognitive dysfunction and the underlying mechanisms.
Autophagy is a programmed cell death (PCD) that digests useless cellular components for keeping cell homeostasis [20]. Autophagic disorder plays a crucial role in neurologi- cal dysfunction, like Parkinson’s disease and Alzheimer’s disease [21]. Strikingly, we have demonstrated that exces- sive autophagy in the hippocampus plays an important role in cognitive impairment caused by SD [22]. Furthermore, recent direct experimental data indicated that suppressing excessive autophagy protects hippocampal neurons under sleep deprivation [23]. It has been confirmed that H2S allevi- ates cerebral ischemia–reperfusion injury through suppress- ing excessive autophagy [24, 25]. Therefore, we explore whether H2S is through suppressing excessive autophagy to alleviate SD-induced cognitive impairment.
Sirt-1 (Silent information regulator 1) is an important nicotinamide-adenine dinucleotide (NAD)-dependent his- tone deacetylase in mammals [26]. A large number of stud- ies have proved that Sirt-1 functions as a neuroprotective molecule and is essential for normal learning and memory function and synaptic plasticity [27–30]. Recently, it has also been reported that Sirt-1 inhibits corticosterone-induced autophagy [31]. Interestingly, our previous study demon- strated that H2S alleviates SD-caused oxidative stress, ER stress, and apoptosis in the hippocampus by upregulation of Sirt-1 [32]. Therefore, we investigated whether Sirt-1 medi- ates the antagonistic role of H2S in SD-caused cognitive by regulating excessive autophagy.
In the present work, we found the suppressive effects of H2S on the cognitive disorder and the excessive hippocampal autophagy in the SD-exposed rats and the inhibitory role of Sirtinol, the inhibitor of Sirt-1, in these above suppressive effects of H2S. Those results suggested that H2S prevents SD-induced cognitive dysfunction by suppressing the hip- pocampal excessive autophagy, which is mediated by Sirt-1.

Materials and Methods
Chemicals

Sodium hydrosulfide (NaHS, a donor of H2S) and Sirti- nol (an inhibitor of Sirt-1) were obtained from Sigma (St. Louis, MO, USA). The primary antibodies of Beclin1, and p62 were obtained from Cell Signaling Technology (Dan- vers, MA, United States). The β-actin antibody and Goat anti- rabbit antibody were obtained from Proteintech (Chi- cago, USA). Bicinchoninic Acid (BCA) Protein Assay Kit was purchased from Beyotime Institute of Biotechnology (Shanghai, China).
Animals

Adult male Wistar rats (200 ± 20 g), used in this experiment was purchased from the SJA Lab Animal Center of Chang- sha (Changsha, China). The rats were housed individually in a room with suitable temperature (25 ± 2) °C, constant humidity, natural illumination, and good ventilation, as well as provided water and food ad libitum to rats. Before the experiment, the rats were adapted to the experimenter and the environment for a week to eliminate the strangeness and reduce the interference and influence of human factors and surrounding environment on the experimental rats. This study was approved by the Animal Use and Protection Com- mittee of the University of South China and was conducted in accordance with the regulations on the Administration of Laboratory Animals promulgated by the State Science and Technology Commission of the People’s Republic of China.
Experimental Design and Schedule

 

In order to explore whether H2S antagonize SD-induced cog- nitive impairment and excessive autophagy, the first batch of Wistar rats were randomly divided into five groups: control group, SD group, SD with low dose (30 μmol/kg) H2S treat- ment group, SD with high dose (100 μmol/kg) H2S treatment group and H2S (100 μmol/kg) treatment group. In order to explore whether blocking Sirt-1 by Sirtinol reverse the pro- tective effect of H2S on SD-induced cognitive impairment and excessive autophagy, the second batch of Wistar rats were randomly divided into five groups: control group, SD

group, SD with H2S (100 μmol/kg) treatment group, SD with H2S (100 μmol/kg) and Sirtinol (10 nmol/kg) treatment group and Sirtinol (10 nmol/kg) group.
Before the test, all rats were habituated to the experi- menter and the laboratory for 1 week. Rats were pre- treated with NaHS (30/100 μmol/kg, ip) for 7 days and then cotreated with SD and/or Sirtinol (10 nmol/d, icv) for 72 h. The Y-maze was performed one day after SD. The NORT was performed 2 days after Y-maze test. The OLT was performed 2 days after NORT. The MWM test was per- formed 2 days after OLT. One day after behavioral testing, rats were deeply anesthetized by intraperitoneal injection of 0.6% pentobarbital sodium, the hippocampal tissues were collected for detecting the level of autophagy.
The doses and frequencies of NaHS administration (30 or 100 μmol/kg/d, 14 days, i.p.) followed the previous research
[33] and our previous work [32, 34]. Sirtinol administration (10 nmol/day, for 72 h, i.c.v.) followed the previous research
[35] and scheme determined in our previous work [32, 34].
Induction of Sleep Deprivation

Modified multiple platform method (MMPM) was used to induce sleep deprivation (SD) [36, 37]. This apparatus was a water tank (170 cm long × 70 cm wide × 50 cm high) con- tained 19 small platforms (6.5 cm diameter) which located 1 cm above the water surface. Rats were placed on the plat- forms and could jump from one platform to another. Rats in each group were placed on the apparatus at 1 week before the experiment for 6 h per day. Once the rat enters rapid eye movement (REM) sleep, losing the whole-body skeletal muscle tone causing the body touch or fall into the water and wake up, resulting in sleep deprivation (SD). During the sleep deprivation period (72 h), the laboratory temperature was kept at (23 ± 2) °C, and the water temperature was con- trolled at (20 ± 2) °C and under a 12:12 h light–dark cycle. In addition, in order to exclude the influence of environmen- tal factors, the control rats were placed on a wide platform (12 cm diameter) in the water tank to allow the rats entered REM sleep without falling into the water.
Intracerebroventricular Injection

The animals were placed in a stereo positioning frame after anesthetizing with 1% sodium pentobarbital (0.04 ml/kg, i.p.). The incision area was trimmed with a sterile surgical scissors. Sirtinol (10 nmol) was injected unilaterally into the ventricle using a 10-μL Hamilton syringe using the follow- ing coordinates: AP: 1.0 mm, R or L: 1.5 mm, at an injection rate of 0.75 μL/min. The needle should be slowly pulled out in half and kept in the place for 2 more minutes before removal to ensure that the drug had been fully delivered.

After operation, all rats should be injected penicillin subcu- taneously for 3 consecutive days.
The Y‑Maze Test

Y-maze is mainly used to test discrimination learning, work- ing memory and reference memory in animals [17, 38]. The Y-maze device consists of three arms of the same size (120°, 55 cm long × 16 cm wide × 20 cm high), which were desig- nated as arm A, B and C, each arm must be painted black to avoid seeing other arms. The apparatus must be clean and symmetrical to eliminate the influence of visual and olfac- tory cues on the results. At the beginning of the experiment, the rats were placed in the center of the Y maze and allowed to explore freely for eight minutes and the total alternat- ing numbers and sequences of each rat entering the three arms were recorded. The body and tail of the rat entered the arm completely was recorded as an effective exploration and the rat entered the three arms sequentially (for example, A/B/C or C/B/A, etc.) was recorded as correct alternating. The following formula is used to calculate the alternating performance: the correct alternating rate = the correct alter- nating number/(alternating total-2). Meanwhile, the total alternating number of rats entering the three arms was used to measure the activity of the rats.
The Novel Object Recognition (NOR) Test

The new object recognition test is a high verification method for hippocampal related cognitive function [17, 38]. The novel object recognition test was carried out according to the method described earlier [39], consisted of three phases: adaption, training and testing phase. In the adaptation phase, rats were placed in an opaque empty square box (50 cm × 50 cm × 60 cm) for 2 days, 5 min per day. And then, during the training phase, the rats were placed into the behavioral box with two identical objects (A and B), meanwhile, to ensure the same distance between the rats and the two objects. Every rat explored in the behavioral box for 5 min, and the time of exploration in the range of 2 cm from each object was recorded respectively (including the squatting object, the front paw on the object, and the nose olfactory object). In the testing phase (for 1 h resting after the training phase), one of the old objects
(B) was replaced with a novel object (C) and the follow- ing experimental steps are the same as the training phase and the observation time is also 5 min. Calculate the total exploration time of the training phase and testing phase and the recognition index (RI) and discrimination index (DI) of the test phase to determine the animal’s cognitive ability: RI = exploration time of C object/total exploration time, DI = (exploration time of C object − exploration time of A

object)/total exploration time. The effects of animal motor ability and emotional differences on cognitive ability were evaluated by the total exploration time during the training phase (e1) and the testing phase (e2).
Object Location Test

Object location test (OLT) has the same rules as NOR test. The difference between OLT and NOR test is that the explo- ration time measured from two identical objects, but one was placed in a novel location. The recognition index (RI), simi- lar to NOR test, was calculated as the time spent on explor- ing on the object in the new location divided by the total time spent on exploring the two objects. Spatial memory and discrimination were evaluated in this test.
The Morris Water Maze (MWM) Test

The Morris water maze can objectively measure the changes of spatial memory, working memory and spatial discriminability in animals [34, 38, 40]. The apparatus was a circular pool (180 cm diameter, 60 cm high) with a movable submerged platform (12.5 cm diameter) kept
0.5 cm below the water level. The depth of opaque white water mixed with titanium oxide was about 15 cm, the water temperature was controlled at 22 ± 1.0 °C. The pool is divided equally into four quadrants of A, B, C, D. The platform surface was white, so that the rats could not see it. The swimming tracks and experimental data of rats searching for the platform in the experiment were col- lected by TM-Vision Behavioral Experimental System (Techman, Chengdu, China). Here are the experimental phases used in this experiment:
The Acquisition Trial

A quadrant was randomly selected to put the rat into the water facing towards the tank wall, and then recording the time of finding the platform and the swimming route. If the platform is still not be found in the 120 s, guide the animal to the platform and stay for 20 s. Each rat was given 4 trials per day with 20 min interval and continuous training for 5 days.
The Probe Trial

On the second day after the acquisition trial, the platform was removed and the rats were placed in water from the opposite side of the target quadrant (the quadrant where the platform was originally placed) for 120 s. The number of times the rats entered the target quadrant and the dura- tion of stay were recorded as an index to detect spatial memory.

The Visible Platform Trial

After the probe trial, the platform was risen to above the water level for 2 cm, and the experimental procedure was the same as the acquisition trial. The purpose of this procedure was to exclude the effects of non-cognitive factors such as motor ability and vision on the experiment.
Western Blot Analysis

First, the supernatant of the collected tissue homogen- ate was used to quantify the total protein concentration by BCA Protein Assay Kit. The protein lysate which mixed with 5 × loading buffer and heated at 100 °C for 5 min was applied to a sodium dodecyl sulfate–polyacrylamide gel for electrophoresis, and then transferred to a PVDF membrane using a wet transfer system. The blots were blocked with 5% skim milk in TBST buffer (50 mmol/L Tris–HCl, pH 7.5, 150 mmol/L NaCl, 0.1% Tween-20) for 2 h and incu- bated with primary antibodies of SYN1, beclin1, and P62 (1:1000) and β-actin (1:2000) overnight at 4 °C. Then, the membranes were washed 3 times for 10 min using TBST and incubated with secondary antibody with horseradish peroxi- dase (1:5000) for 2 h. Finally, the protein membranes were washed in the same manner and the bands were visualized by an enhanced chemiluminescence system (BeyoECL Plus kit, Beyotime, P0018). The immunoblot signal was analyzed by AlphaImage 2200 software; meanwhile, β-actin was set as a loading control.
Transmission Electron Microscopy (TEM)

Transmission electron microscopy was used to observe the ultrastructural changes in the hippocampus of rats. The col- lected hippocampus tissues were cut into 1 mm3 pieces, obtained in 2% glutaraldehyde in 0.1 M PBS solution imme- diately for fixation at 4 °C overnight. Next, these tissues rinsed in 0.1 M PBS pH 7.4 for 20 min and fixed in 1% Osmium tetroxide in 0.1 M PBS for 2 h. After dehydrated with gradient ethanol and infiltrated with epon–acetone, the specimens were embedded in epoxy resin. After incubation at 70 °C vacuum oven for 2 days, epoxy resin blocks are ready for sectioning. Next, the pieces were cut into ultrathin Sects. (70 nm) with an ultramicrotome (Leica, Germany, EM UC6) and double stain with 1% uranyl acetate and alkaline lead citrate together. Finally, the microphotographs were obtained by transmission electron microscopy (JEOL, Japan, JEM 1230).
Statistical Analysis

Statistical analysis of all data was performed by SPSS 20 software (SPSS, Chicago, IL, United States). Data

are displayed as the mean ± SD. The significance of intergroup differences was evaluated by one-way ANOVA analysis. Statistical significance was indicated at p < 0.05.

Results
H2S Improves SD‑Induced Working Memory Impairment in Y‑Maze Test, Which Is Abolished by Inhibition of Sirt‑1

Y-maze test was subjected to detect whether the working memory impairment of SD-exposed rats is reversed by H2S treatment and the mediatory role of Sirt-1. We found that the alternation performance in the SD-exposed rats was signifi- cantly decrease, but notably reversed by NaHS (30 μmol/ kg, 100 μmol/kg) treatment . On the condition that the alternation performance in the rat without SD was not impacted by NaHS (100 μmol/kg) treated alone (1a). Our previous study demonstrated that H2S up-regulates the expression of Sirt-1 in the hippocampus of SD-exposing rats [32]. In order to prove the mediatory role of Sirt-1 in the protection of H2S against SD-induced working memory impairment, we explored whether Sirtinol, the inhibitor of Sirt-1, reverses the protective effect of H2S on working memory impairment in sleep-deprived rats. In the Y-maze test, after treatment with Sirtinol, the correct alternation performance is reduced in the rats cotreated with NaHS and SD ( 1a). There was no significant difference in total arm entries among the seven groups ( 1b). These data indicated that H2S combats SD-caused working memory impairment via Sirt-1.

H2S Ameliorates SD‑Induced Cognitive Dysfunction in the Novel Object Recognition Test, Which Is Reversed by Inhibition of Sirt‑1

Next, the new object recognition test was subjected to detect whether SD-induced cognitive impairment is reversed by H2S treatment and the mediatory role of Sirt-1. In this test, there is a precipitous loss of recognition index in the SD- exposed rats. On the contrary, NaHS restored the loss of rec- ognition index in the SD-exposed rats (. 2a). In addition, the discrimination index even decreased to a negative value after sleep deprivation, which was significantly recovered after H2S treatment during the test period ( 2b). How- ever, the recovering role of NaHS in the decreased recogni- tion index of SD-exposed rats was abolished under Sirtinol treatment ( 2a). 2b also shows that after blocking Sirt-1, the discrimination index of rats cotreated with SD and H2S was decreased to a negative value. The statistic dif- ference was not significant in total object exploration time among the five groups during training period (2c) or testing period ( 2d). These data indicated that H2S ame- liorates SD-induced cognitive dysfunction via Sirt-1.
H2S Improves SD‑Induced Location Memory Deficit in Object Location Test, Which Is Reversed by Inhibition of Sirt‑1

In order to further explore the role of H2S in the decline of location memory after sleep deprivation, we performed an object location test. In this experiment, we found that pretreat- ment with NaHS (30 μmol/kg/d, 100 μmol/kg/d, i.p) signifi- cantly alleviated the decline of recognition index in the SD- exposed rats ( 3a). Similarly, the discrimination index in

 1 Effect of H2S and Sirtinol on SD-induced working memory impairment of rats in the Y male test. Rats were pretreated with NaHS (30 μmol/kg/d and 100 μmol/kg/d, ip) for 7 days and then cotreated with SD and Sirtinol (10 nmol/d, icv) for 72 h and were performed in the Y male test. The alternation performance (a) and
the total arm entries (b) were recorded. Values were presented as mean ± SD. (n = 10–14). ***P < 0.001, vs control group; ###P < 0.001, vs sleep deprivation alone group; &&&P < 0.001 vs sleep deprivation group treated with NaHS (100 μmol/kg, i.p.)

 2 Effect of H2S and Sirtinol on SD-induced cognitive dysfunc- tion of rats in the novel object recognition test. Rats were pretreated with NaHS (30 μmol/kg/d and 100 μmol/kg/d, ip) for 7 days and then cotreated with SD and Sirtinol (10 nmol/d, icv) for 72 h and tested in the novel object recognition test. The recognition index (a) and discrimination index (b) in the test period as well as the total
object exploration time in the training period (c) or in the test period
(d) were recorded. Values were presented as mean ± SD (n = 9–11).
***P < 0.001, vs control group; ##P < 0.01, vs sleep deprivation alone group; &P < 0.05, vs. sleep deprivation group treated with NaHS (100 μmol/kg, i.p.)the SD-exposed rats was decreased linearly to a negative value, which was recovered by NaHS treatment  Next, we explored whether Sirtinol reverses the protection of H2S against the location memory deficit in the sleep-deprived rats in the object location test. After treatment with Sirtinol, the values of recognition index ( 3a) and discrimination index ( 3b) in the rats cotreated with NaHS and SD were decreased. However, there were no significant differences between groups in the total number of object exploration during training period (3c) or testing period (3d). These results indicated that H2S improves the SD-caused location memory deficit via Sirt-1.

H2S Improves SD‑Induced Spatial Learning
and Memory Disorder in the Morris Water Maze Test, Which Is Reversed by Inhibition of Sirt‑1

The Morris water maze test also was used to prove whether H2S protects against the spatial learning and memory dysfunction

in SD-exposed rats. The track of the sleep-deprived rats on the fifth day of the acquisition trial phase is more complicated than that of the control group, which is simplified after NaHS treatment (4a), indicating that NaHS improves the spatial learning of SD-exposed rats. On the 5th day of the acquisi- tion trial phase, the escape periods of sleep-deprived rats were significantly longer than that of normal rats and significantly shortened after treatment with 100 μmol/kg of NaHS ( 4b). In the probe trial phase, the times of crossing platform ( 4c) and the percentage of time in the target quadrant ( 4d) were reduced in the SD-exposed rats, which were restored by treat- ment with NaHS, indicating that H2S improves the spatial memory of SD-exposed rats. We further explored the effects of Sirtinol on the protections of H2S against the spatial learn- ing and memory impairments in sleep-deprived rats using the Morris water maze test. On the fifth day of acquisition trial phase, Sirtinol reversed the simplified swimming route (4a) and the shortened latency time  in the rats

3 Effect of H2S and Sirtinol on SD-induced location memory dysfunction of rats in the object location test. Rats were pretreated with NaHS (30 μmol/kg/d and 100 μmol/kg/d, ip) for 7 days and then cotreated with SD and Sirtinol (10 nmol/d, icv) for 72 h. After that, rats were tested in the object location test. The recognition index (a) and discrimination index (b) in the test period as well as the total

object exploration time in the training period (c) or in the test period
(d) were recorded. Values were presented as mean ± SD (n = 7–11).
**P < 0.01, vs control group; ##P < 0.01, ###P < 0.001, vs sleep dep- rivation alone group; &P < 0.05, vs. sleep deprivation group treated with NaHS (100 μmol/kg, i.p.)cotreatment with NaHS with SD. In the probe trial phase, the times of crossing platform (. 4c) and the percentage of time in the target quadrant ( 4d) in the rats cotreatment with NaHS and SD were reduced after treatment with Sirtinol. In the visible platform trial phase, there was no significant differ- ence both in the latency to reach the platform ( 4e) and in the average swimming speed ( 4f) of the rats in all seven groups, suggesting that the factors affecting their visual per- ception and swimming capability were excluded. These results indicated that H2S improves the SD-induced spatial learning and memory disorders via Sirt-1.

H2S Protects Against SD‑Induced Hippocampal Excessive Autophagy, Which Is Reversed
by Inhibition of Sirt‑1

The hippocampal excessive autophagy contributes to SD- exerted cognitive impairment [22]. We next investigated

whether H2S inhibits the excessive autophagy in the hip- pocampus of SD-exposed rats. Autophagosomes were accumulated in the hippocampus of sleep-deprived rats, which were significantly reduced by treatment with NaHS (30 μmol/kg or 100 μmol/kg, i.p.) ( 5a). The expres- sion of Beclin1 was increased ( 5b) and the expres- sion of p62 was decreased ( 5c) in the hippocampus of sleep-deprived rats, which were reversed by NaHS (30 μmol/kg/d and 100 μmol/kg/d, i.p.). NaHS alone did not alter the number of autophagosomes and the expres- sions of Beclin1 and p62 in the hippocampus of normal rats. Next, to determine whether Sirt-1 is essential for H2S to inhibit excessive hippocampal autophagy in sleep- deprived rats, we examined whether Sirtinol blocks the protection of NaHS against the excessive autophagy in the hippocampus of sleep-deprived rats. Sirtinol sig- nificantly reversed the effects of NaHS by showing the increases in the number of autophagosomes (5a) and
4 Effects of H2S and Sirtinol on SD-induced spatial learning and memory disorders in rats. Rats were pretreated with NaHS (30 μmol/kg/d and 100 μmol/kg/d, ip) for 7 days and then cotreated with SD and Sirtinol (10 nmol/d, icv) for
72 h and tested in the Morris Water Maze. (a) The representa- tive swimming tracks of rats searching for the underwater platform at the 1st and 5th train- ing days; (b) The escape periods traveled to find the platform during five days in the acquisi- tion phase; (c–d) The number
of times that the rats crossed the platform (c) and the proportion- ality of swimming time in target quadrant (d) in probe trial were analyzed; (e–f) The latency
to reach the platform (e) and the average swimming speed to reach the platform (f) in the visible platform test. Values were presented as mean ± SD
(n = 9–10). *P < 0.05, **P < 0.01,
vs control group; #P < 0.05, vs sleep deprivation alone group; &P < 0.05, vs sleep depriva- tion group treated with NaHS (100 μmol/kg, i.p.)the expression of beclin1 ( 5d) as well as the decrease in the expression of P62 ( in the hippocampus of rats cotreatment with NaHS and SD. These data indicate that H2S antagonizes SD-induced hippocampal excessive autophagy via Sirt-1.
Discussion
The purpose of this study was to investigate the antag- onistic role of H2S in SD-caused cognitive impair- ment and the underlying mechanisms. In the present study, we for the first time found that H2S attenuates
5 Effects of SD, H2S and Sirtinol on the autophagy in the hippocampus of rats. Rats were pretreated with NaHS (30 μmol/ kg/d and 100 μmol/kg/d, ip) for 7 days and then cotreated with SD and Sirtinol (10 nmol/d, icv) for 72 h and the hippocampus of rat was separated. Autophagic vacuoles [(a), magnifica-
tion × 5000; scale bar = 5 μm] were observed under trans- mission electron microscope. Arrows indicate autolysosome- like vesicles in the cytoplasm. The expressions of Beclin-1 (b, d) and P62 (c, e) in the hip- pocampus of rats were detected by Western blot using anti- Beclin-1 and -P62 antibody,
respectively. β-actin was used as loading control. Data were pre- sented as mean ± SD (n = 3–5).
**P < 0.01, ***P < 0.001, vs con- trol group; #P < 0.05, ##P < 0.01, ###P < 0.001, vs sleep depriva- tion alone group; &P < 0.05, vs sleep deprivation group treated with NaHS (100 μmol/kg, i.p.)cognitive dysfunctions and inhibits the excessive hippocam- pal autophagy in the SD-exposed rats. Furthermore, Sirtinol, a special inhibitor of Sirt-1, reversed the antagonistic role of H2S in SD-caused cognitive impairment and excessive hippocampal autophagy. We have previously confirmed that H2S upregulates the expression of Sirt-1 in the hippocam- pus of SD-exposed rats [32]. Together, these data make it reasonable to conclude that H2S antagonizes SD-induced cognitive impairment by inhibiting excessive autophagy via upregulation of hippocampal Sirt-1.

Increasing studies demonstrated the damage of SD to cognitive function in multiple ways [41]. Therefore, the

prevention and treatment of SD-induced cognitive impair- ment is an emerging area of research. The hippocampus is an important regulatory part of the limbic system of the brain, closely related to emotional regulation, learning, memory, and cognitive function [42, 43]. It has been proved that structural and function changes in hippocampus promote neurological pathology [44]. Our previous study showed that the reduction of hippocampal endogenous H2S production involves in SD-induced cognitive dysfunction [22], which suggests that exogenous H2S supplementary has a poten- tial to resist SD-induced cognitive dysfunction. To explore the effect of H2S supplementary on SD-induced cognitive

dysfunction, Y-maze, Novel object recognition (NOR), object location, and Morris water maze (MWM) tests were used. These results are in agreement with previous evidence in rats showing that sleep deprivation leads to learning and memory impairment [45–48]. However, after treatment with H2S, SD-induced cognitive dysfunction was reversed. It has been demonstrated that H2S prevents the cognitive dysfunc- tion induced by homocysteine [34], chronic restrain stress [38], diabetes [17]. Therefore, it is reasonable to assume that H2S has a promising future for the prevention and treatment of cognitive dysfunction induced SD.
Autophagy is the major intracellular degradation system, maintaining metabolic balance via digesting misfolded pro- teins and dysfunctional organelles [20], which play a positive role in maintaining neuronal homeostasis [49]. Importantly, increasing evidence confirm that excessive hippocampal autophagy contributes to cognitive dysfunction [50–52]. In this study, we found that increased autophagosome and Beclin1 as well as decreased p62 in the hippocampus of SD- exposed rats were reversed by H2S treatment, which indi- cated that H2S inhibits SD-induced excessive hippocampal autophagy. Therefore, we suggest that suppressing excessive hippocampal autophagy plays an important role in the antag- onistic action of H2S in SD-caused cognitive impairment.
Sirt-1 protects against aging-induced damage and extends lifespan [53]. Interestingly, recent studies have found that Sirt-1 in the cerebral cortex, cerebellum and hippocampus is essential for cognitive function [29, 54]. Sirt-1 not only mediates resveratrol-prevented cognitive deficits induced by chronic unpredictable mild stress [55], but also mediates AMPKα1 to improve postoperative cognitive dysfunction [56]. In addition, it has been asserted the inhibitory role of Sirt-1 in autophagy [38]. We have previously confirmed that H2S upregulates the expression of Sirt-1 in the hippocampus of SD-exposed rats [32]. Therefore, we believed that Sirt-1 is an effector in keep the homeostasis of autophagy and is a target for the function of H2S. In this study, we found that the antagonistic actions of H2S in SD-caused cognitive impair- ment and excessive hippocampal autophagy were abolished by Sirtinol, the inhibitor of Sirt-1. Together with our previ- ous finding that H2S upregulates the expression of Sirt-1 protein in the hippocampus of rats [57], it is reasonable to conclude that the protective effects of H2S on SD-induced cognitive impairment and excessive hippocampal autophagy are in a Sirt-1-dependent manner.
Totally, our results show that H2S prevents the cogni-
tive disorder and suppresses the excessive hippocampal autophagy in the SD-exposed rats and that inhibited Sirt-1 abolished these beneficial effects of H2S. These data, together with the previous finding that H2S upregulates the expression of Sirt-1 in the hippocampus of SD-exposed rats [32], raise the exciting possibility that H2S suppresses the excessive hippocampal autophagy to attenuate SD-induced

cognitive dysfunction in a Sirt-1-dependent manner. The present findings for the first time suggest that H2S hold promise as potential therapeutic agents for the treatment of SD-exerted cognitive impairment.
Acknowledgements This work was supported by National Natural Sci- ence Foundation of China (81771178), Natural Science Foundation of Hunan province (2019JJ80101), and the Major Research Topics of the Health Commission of Hunan province (20201911).

Data Availability The datasets used or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Conflict of interest The authors declare that there are no conflicts of interest associated with this study.

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