The Role of LincRNA-EPS/Sirt1/Autophagy Pathway in the Neuroprotection Process by Hydrogen against OGD/R-Induced Hippocampal HT22 Cells Injury

Cerebral ischemia/reperfusion (CI/R) injury causes high disability and mortality. Hydrogen (H2) enhances tolerance to an announced ischemic event; however, the therapeutic targets for the effective treatment of CI/R injury remain uncertain. Long non-coding RNA lincRNA-erythroid prosurvival (EPS) (lincRNA-EPS) regulate various biological processes, but their involvement in the effects of H2 and their associated underlying mechanisms still needs clarification. Herein, we examine the function of the lincRNA-EPS/Sirt1/autophagy pathway in the neuroprotection of H2 against CI/R injury. HT22 cells and an oxygen-glucose deprivation/reoxygenation (OGD/R) model were used to mimic CI/R injury in vitro. H2, 3-MA (an autophagy inhibitor), and RAPA (an autophagy agonist) were then administered, respectively. Autophagy, neuro-proinflammation, and apoptosis were evaluated by Western blot, enzyme-linked immunosorbent assay, immunofluorescence staining, real-time PCR, and flow cytometry. The results demonstrated that H2 attenuated HT22 cell injury, which would be confirmed by the improved cell survival rate and decreased levels of lactate dehydrogenase. Furthermore, H2 remarkably improved cell injury after OGD/R insult via decreasing pro-inflammatory factors, as well as suppressing apoptosis. Intriguingly, the protection of H2 against neuronal OGD/R injury was abolished by rapamycin. Importantly, the ability of H2 to promote lincRNA-EPS and Sirt1 expression and inhibit autophagy were abrogated by the siRNA-lincRNA-EPS. Taken together, the findings proved that neuronal cell injury caused by OGD/R is efficiently prevented by H2 via modulating lincRNA-EPS/Sirt1/autophagy-dependent pathway. It was hinted that lincRNA-EPS might be a potential target for the H2 treatment of CI/R injury.


Introduction
Cerebral ischemic injury is a major contributor to death or different degrees of disability worldwide. Cerebral ischemic injury may result from several different events, including external forces and degradation of the blood vessels, as well as thrombotic or thromboembolic arterial occlusions, etc. [1][2][3][4]. Clinically, cardiopulmonary resuscitation from cardiac arrest is among the leading contributors to cerebral ischemia-reperfusion (CI/R) injury. The timely restoration of blood flow is the key to treating cerebral ischemia. However, reperfusion after ischemia has been accompanied by a series of pathological reactions, which could cause permanent damage to brain tissue, and these reactions include the production treatment strategies that target autophagy as a mechanism for treating CI/R injuries show great promise.
To further understand how H 2 protects neurons from CI/R damage, we conducted an in vitro investigation to examine the function of the lincRNA-EPS/Sirt1/autophagy pathway.

Cell Culture
HT22 cell, an immortalized mouse hippocampal neuronal cell line, was subcloned from parent HT4 cells originally immortalized from cultures of primary mouse hippocampal neurons [33]. HT22 cell was obtained from the Laboratory of Anesthesiology, Southwest Medical University (Luzhou, China). HT22 cells were cultured in a humid incubator at 37 • C containing 5% CO 2 and 95% air environment in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 100 U penicillin/streptomycin.

OGD/R Injury Model Development
The induction of OGD/R injury in HT22 cells was performed as reported in previous studies [32,34,35]. After being rinsed thrice in phosphate-buffered saline (PBS), the cells were cultured in a serum-free, glucose-free DMEM culture. Immediately after harvesting, the cells were introduced in a tri-gas incubator (Eppendorf, Hamburg, Germany) for 24 h at 37 • C for 2-, 4-, 6-, 8, or 12 h with 4% N 2 /5% CO 2 /1% O 2 . Following these procedures, the cells were returned to an oxygenated, glucose-containing DMEM under normoxic conditions for 24 h (reoxygenation). In addition, 3-methyladenine (3-MA) or rapamycin (RAPA) was added into the medium at the beginning of reoxygenation and left until the end of reoxygenation.

Synthesis of a Medium Enriched with Hydrogen
The procedure for creating a hydrogen-rich medium has already been detailed [36]. In brief, hydrogen was dissolved in a DMEM at a pressure of 0.4 MPa. Then, the concentration of hydrogen in the solution was determined using a hydrogen electrode, and when the H 2 concentration reached 0.6 mmol/L, the preparation of a medium enriched with hydrogen was completed. The medium was freshly produced to guarantee high H 2 concentration. In the H 2 group, a hydrogen-rich medium was used to culture the cells, rather than the normal medium at the beginning of the reoxygenation.

Experimental Design
The HT22 cells were classified for the following tests in the current investigation. To find out an optimal OGD time, these six groups of cells were created by a random sampling process: (1) control (Con) group-except for not performing OGD/R operation, all other operational steps were the same as those in the OGD/R group; (2) OGD-2h/R group-OGD for 2 h plus 24 h of reoxygenation; (3) OGD-4h/R group-OGD for 4 h plus 24 h of reoxygenation; (4) OGD-6h/R group-OGD for 6hrs plus 24 h of reoxygenation; (5) OGD-8h/R group-OGD for 8 h plus 24 h of reoxygenation; (6) OGD-12h/R group-OGD for 12 h plus 24 h of reoxygenation.
The cells were randomized into the following 4 groups to examine the protective properties of H 2 on OGD/R injury in HT22 cells: (1) control (Con) group; (2) hydrogen (Con+ H 2 ) group-except for not performing OGD/R operation, all other operational steps were as same as those in the OGD/R+ H 2 group; (3) OGD/R group; (4) OGD/R+ H 2 -cells were cultured in a medium enriched with H 2 rather than the normal medium at the beginning of the reoxygenation, and all other operational steps were the same as those in the OGD/R group.
To additionally examine the underlying protective mechanism of H 2 , we utilized 3-MA and RAPA to inhibit autophagy in cells and used small interfering RNAs (siRNAs) to knock down intracellular lincRNA-EPS. First, to examine the involvement of autophagy in the protective function of H 2 on OGD/R injury in HT22 cells, the cells were randomized into 6 groups as indicated: (1) OGD/R group; (2) OGD/R+ H 2 group; (3) OGD/R+3-MA group-10 mM 3-MA (J&K Scientific, Beijing, China) was introduced into the normal medium at the beginning of the reoxygenation; (4) OGD/R+ H 2 +3-MA group-10 mM 3-MA was added into the hydrogen-rich medium at the beginning of the reoxygenation; (5) OGD/R+RAPA group-500 nM RAPA (Absin, Shanghai, China) was added into the normal medium at the beginning of the reoxygenation; (6) OGD/R+ H 2 +RAPA group-500 nM RAPA was added into the H 2 -rich medium at the beginning of the reoxygenation. Then, to confirm whether H 2 could exert a cytoprotective effect by activating intracellular lincRNA-EPS to inhibit autophagy following OGD/R injury, the cells were randomized into the following six groups: (1) OGD/R group; (2) OGD/R+ H 2 group; (3) OGD/R+siRNA-NC group-siRNA-NC was transfected into the cells 48hrs before OGD/R.; (4) OGD/R+siRNA group-siRNA-lincRNA-EPS was used to transfect the cells 48hrs before OGD/R; (5) OGD/R+ H 2 +siRNA-NC group; (6) OGD/R+ H 2 +siRNA group.

Cell Viability and Cytotoxicity Assays
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test was conducted to determine whether or not HT22 cells were viable [37]. In total, 200 µL of growth medium was used to seed cells into 96-well plates (5 × 10 3 cells/well), after which they were incubated overnight and treated according to different experimental groups. A 24 h reoxygenation period was followed by the addition of 10 µL of MTT solution per well and a 4-h incubation period at 37 • C. A volume of 100 µL of dimethyl sulfoxide (DMSO) was added to the culture after the media had been gently aspirated. Afterward, spectrophotometer readings were taken at 570 nm to measure each well's optical density (OD).
Using a commercially available kit (Jiancheng Bioengineering Institute, Nanjing, China), a lactate dehydrogenase (LDH) test was conducted to identify the HT22 cells' membrane permeability following the directions stipulated by the manufacturer.

Cell Apoptosis Assay
For the analysis of cell apoptosis, an Annexin V-FITC/PI kit (BD Biosciences, Franklin Lakes, NJ, USA) was utilized. In brief, following the seeding of 6-well plates with HT22 cells (1 × 10 5 cells/well), they were cultured for a full night, and treated according to different experimental groups. After collecting the cells using trypsin without EDTA, they were rinsed thrice using PBS. Then, following the resuspension of the cells in binding buffer, they were stained with 5 µL of Annexin-V FITC and propidium iodide (PI), and then they were subjected to incubation for 15 min in darkness at room temperature (RT), as instructed by the manufacturer. Finally, the apoptotic rate was determined by a flow cytometer (BD Biosciences) [38].

Detection of Inflammatory Factors
Cell culture supernatant was collected after OGD/R. Then, The enzyme-linked immunosorbent assay (ELISA) kits (Meilian Co., Ltd., Wuhan, China) were used to measure the levels of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) that were present in the supernatant of cell cultures in compliance with the directions provided by the manufacturer as described previously [39].

Tandem mCherry-EGFP-LC3 Immunofluorescence
Lenti-mCherry-EGFP-LC3B (Beyotime Institute of Biotechnology, Shanghai, China) was introduced into HT22 cells via transfection [40]. After puromycin selection for positive transfection, EGFP-positive HT22 cells were visualized by a microscope, and EGFP-positive colonies were harvested and expanded for subsequent experiments. Before treatment, HT22-mCherry-EGFP-LC3B cells were grown throughout the night on glass coverslips in a DMEM containing 10% FBS. After the treatment, a 4% paraformaldehyde (PFA) solution was applied to fix the cells on the coverslips for half an hour in darkness at RT before rinsing them thrice in PBS, labeling them with 4 ,6-diamidino-2-phenylindole (DAPI) for 6-8 min, and rinsing again using PBS. An upright fluorescent microscope (Nikon, Tokyo, Japan) was used to observe the cells' EGFP and mCherry fluorescence. The nucleus was visualized with the help of DAPI staining. The ratio of the fluorescence of mCherry+ puncta to the fluorescence of EGFP+ puncta was used as the autophagic index. Cells were photographed and scored in a blind manner utilizing the ImageJ program (National Institutes of Health, Bethesda, MD, USA). More than 50 cells were counted for each condition.

Real-Time Polymerase Chain Reaction (RT-PCR)
Total RNA was isolated from the HT22 cells using the TRIzol reagent (Tiangen, Beijing, China), as per the package directions. Afterward, we utilized a HiScrip III All-in-one RT SuperMix Perfect for qPCR kit (Vazyme Biotech Co., Ltd., Nanjing, China) to reverse transcribe 1 µg of the extracted total RNA. RT-PCR was conducted utilizing a Taq Pro Universal SYBR kit (Vazyme Biotech Co., Ltd.) in a real-time PCR system (Roche, Basel, Switzerland). An initial incubation at 95 • C for 15 min was accompanied by 5 cycles of incubation at 95 • C for 10 s and at 60 • C for 32 s to accomplish the amplification. The procedure started with a 30 s denaturation at 95 • C, followed by 40 cycles of 10 s denaturation at 95 • C and 30 s annealing at 60 • C. Melting curve analysis was conducted at 95 • C for 15 s, and annealing at 60 • C for 60 s and at 95 • C for 15 s as directed by the manufacturer. Table 1 displays the RT-PCR primer sequences. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was chosen to serve as the internal reference. Additionally, the 2 −∆∆CT method was utilized to measure the relative mRNA levels. Target gene mRNA expression was represented as a fold-change compared with the Con or OGD/R group [39].

Western Blot Assay
Following the reoxygenation process, the HT22 cells were collected for analysis. RIPA lysis solution (Beyotime Institute of Biotechnology) containing 1% phenylmethylsulfonyl fluoride (PMSF) was utilized to lyse these cells for half an hour on ice. Cell lysates were centrifuged at a rate of 14,000× g for 10 min at 4 • C, and the protein concentration of the supernatant was quantified using the bicinchoninic acid assay (BCA) kit (Beyotime Institute of Biotechnology). In addition, 20 µg of protein was loaded, electrophoresed on the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel, transferred to a polyvinylidene fluoride (PVDF) membrane (Amersham Biosciences, NJ, USA) or a nitrocellulose membrane (Beyotime Institute of Biotechnology), blocked with 5% skim milk for 1 h, and probed at 4 • C overnight with the following primary antibodies: anti-Sirt1 (dilution, 1:1000; Cell Signaling Technology (CST), Danvers, MA, USA), anti-Beclin1 (dilution, 1:1000; CST), anti-LC3-II (dilution, 1:1000; CST), and anti-β-actin (dilution, 1:3000; Proteintech, Rosemont, IL, USA). The next step involved incubating the sample for 1 h at 37 • C with a secondary antibody conjugated to horseradish peroxidase (dilution, 1:5000; Absin, Shanghai, China). Eventually, the membrane was subjected to enhanced chemiluminescence (ECL), after which the immunoreactive bands were evaluated with the use of the ImageJ 1.31 program (National Institutes of Health). The data were normalized using beta-actin as the internal control [39].

Statistical Analysis
The analyses of all statistical data were conducted with the use of GraphPad Prism 8.3 statistical software (GraphPad Software Inc., San Diego, CA, USA). Means ± standard deviations (SD) were used to present all data. One-way analysis of variance (ANOVA) and Tukey's post-hoc test were utilized for making comparisons. A p < 0.05 denoted the significance criterion.

The Damage of HT22 Cells Was Aggravated after OGD/R
The survival rate of the HT22 cells gradually decreased with an increase in the duration of OGD treatment, while the amount of LDH produced increased. It was discovered that each OGD/R group had significantly worse HT22 cell injury than the Con group. Based on past research and the fact that only approximately half as many HT22 cells survived in the OGD-6h/R group as did in the Con group, we decided to use the OGD-6h/R group for our subsequent experiments to allow for moderate damage to HT22 cells ( Figure 1).

H 2 Protected HT22 Cells from OGD/R Injury
To determine whether H 2 could protect HT22 cells against damage elicited by OGD/R, the cells were exposed to the hydrogen-rich medium at the beginning of the reoxygenation. When compared with the Con group, the cell survival rate in the OGD/R group was reduced, while the LDH level, the levels of pro-inflammatory factors (TNF-α and IL-1β), and the cellular apoptotic rate were elevated. Additionally, the cell survival rate in the OGD/R+ H 2 group increased, while the LDH level, the levels of pro-inflammatory factors (TNF-α and IL-1β), and the cell apoptosis rate decreased relative to those of the Con group ( Figure 2). Notably, the variation between the Con group and the Con+ H 2 group was insignificant.

H2 Protected HT22 Cells from OGD/R Injury
To determine whether H2 could protect HT22 cells against damage elicited b OGD/R, the cells were exposed to the hydrogen−rich medium at the beginning of the re oxygenation. When compared with the Con group, the cell survival rate in the OGD/R The release of LDH in each group. Statistics were reported using a mean ± SD format. A minimum of three separate repetitions of the experiments yielded the same results. * p < 0.05, ** p < 0.01 vs. Con group.

H 2 Upregulated lincRNA-EPS and Sirt1, and Inhibited Autophagy
The possible protective mechanism of H 2 against neuronal OGD/R injury was explored. We discovered that the lincRNA-EPS as well as both Beclin-1 and LC3-II mRNA and protein levels increased remarkably, while the Sirt1 protein and mRNA levels were decreased in the OGD/R group in contrast to those in the Con group. Meanwhile, H 2 upregulated the lincRNA-EPS and Sirt1 mRNA and protein levels while decreasing the Beclin-1 and LC3-II mRNA and protein levels in the OGD/R damage group relative to the Con group ( Figure 3). These data indicated that H 2 could improve neuronal OGD/R damage by suppressing autophagy and upregulating the lincRNA-EPS and Sirt1. The data were displayed as mean ± SD. Three independent runs of the experiments yielded similar results. ** p < 0.01 vs. Con group. ## p < 0.01 vs. OGD/R group.

H2 Upregulated lincRNA−EPS and Sirt1, and Inhibited Autophagy
The possible protective mechanism of H2 against neuronal OGD/R injury was explored. We discovered that the lincRNA−EPS as well as both Beclin−1 and LC3−II mRNA and protein levels increased remarkably, while the Sirt1 protein and mRNA levels were decreased in the OGD/R group in contrast to those in the Con group. Meanwhile, H2 upregulated the lincRNA−EPS and Sirt1 mRNA and protein levels while decreasing the Beclin−1 and LC3−II mRNA and protein levels in the OGD/R damage group relative to the Con group ( Figure 3). These data indicated that H2 could improve neuronal OGD/R damage by suppressing autophagy and upregulating the lincRNA−EPS and Sirt1.

H 2 Protected Neurons against OGD/R Injury by Suppressing Autophagy
We further explored the underlying protective mechanism of H 2 using 3-MA and RAPA. First, we used some methods to detect neuronal damage. Figure 4 illustrates that the cell survival rate was higher in the OGD/R+ H 2 group compared to that in the OGD/R+ H 2 +RAPA group. Meanwhile, H 2 decreased the levels of LDH and pro-inflammatory factors in the medium, while attenuating the cell apoptosis rate, which could be abolished by RAPA during OGD/R.

H2 Protected Neurons against OGD/R Injury by Suppressing Autophagy
We further explored the underlying protective mechanism of H2 using 3−MA and RAPA. First, we used some methods to detect neuronal damage. Figure 4 illustrates that the cell survival rate was higher in the OGD/R+ H2 group compared to that in the OGD/R+ H2+RAPA group. Meanwhile, H2 decreased the levels of LDH and pro−inflammatory factors in the medium, while attenuating the cell apoptosis rate, which could be abolished by RAPA during OGD/R. Beclin1 and LC3-II protein and mRNA levels were then detected by Western blot analysis and RT-PCR. As illustrated in Figure 5, compared with the OGD/R group, the protein and mRNA levels of Beclin1 and LC3-II were lowered by H 2 and were further reduced by 3-MA, while they were upregulated by RAPA during OGD/R. Beclin1 and LC3−II protein and mRNA levels were then detected by Western blot analysis and RT−PCR. As illustrated in Figure 5, compared with the OGD/R group, the protein and mRNA levels of Beclin1 and LC3−II were lowered by H2 and were further reduced by 3−MA, while they were upregulated by RAPA during OGD/R.   Beclin1 and LC3−II protein and mRNA levels were then detected by Western blot analysis and RT−PCR. As illustrated in Figure 5, compared with the OGD/R group, the protein and mRNA levels of Beclin1 and LC3−II were lowered by H2 and were further reduced by 3−MA, while they were upregulated by RAPA during OGD/R.

The Changes of Autophagic Flux after OGD/R Injury
We began by transfecting HT22 cells with mCherry-EGFP-LC3B before triggering autophagy, then we examined the cell images to determine the autophagic flux alterations. According to the instructions, following the autophagosome-lysosome fusion, EGFP was secreted from mCherry-EGFP-LC3B and degraded in the lysosomes. Changes in fluorescence color from green-red (yellow) to red indicate the production of autolysosomes, a loss in the EGFP signal (green), and a reservation of the mCherry signal (red); these fluctuations are used to measure the autophagic flux. In our research, we used DAPI staining to visualize the nucleus. The results showed that H 2 inhibited autophagy in the HT22 cells. Autophagy in the HT22 cells was remarkably enhanced after treatment with RAPA, and this effect was further amplified when RAPA was combined with H 2 (Figure 6).
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The Changes of Autophagic Flux after OGD/R Injury
We began by transfecting HT22 cells with mCherry−EGFP−LC3B before triggering autophagy, then we examined the cell images to determine the autophagic flux alterations. According to the instructions, following the autophagosome−lysosome fusion, EGFP was secreted from mCherry−EGFP−LC3B and degraded in the lysosomes. Changes in fluorescence color from green−red (yellow) to red indicate the production of autolysosomes, a loss in the EGFP signal (green), and a reservation of the mCherry signal (red); these fluctuations are used to measure the autophagic flux. In our research, we used DAPI staining to visualize the nucleus. The results showed that H2 inhibited autophagy in the HT22 cells. Autophagy in the HT22 cells was remarkably enhanced after treatment with RAPA, and this effect was further amplified when RAPA was combined with H2 ( Figure 6). Collectively, based on these findings, H2 protects neurons from OGD/R injury by suppressing autophagy.

LincRNA−EPS Knockdown Abolished the Protection of H2 against Cell Injury
According to the previous results, it was revealed that H2 upregulated lincRNA−EPS and Sirt1 while inhibiting autophagy under OGD/R injury. However, whether H2 could inhibit autophagy and upregulate Sirt1 through activating lincRNA−EPS should be clarified. Thus, to examine the function of lincRNA−EPS in the protective ability of H2 against neuronal OGD/R−induced autophagy and injury, HT22 cells were transfected with siRNA−lincRNA−EPS.
Firstly, the RT−PCR was conducted to examine the knockdown effect of siRNA−lin-cRNA−EPS after transfection. Figure 7A illustrates that in contrast to the siRNA−NC group, the expression of lincRNA−EPS in the other three siRNA groups was significantly downregulated. Among them, the siRNA2−lincRNA−EPS had the most obvious knockdown effect; therefore, we selected siRNA2−lincRNA−EPS for follow−up experiments. We then detected some indicators to evaluate the neuronal injury. As displayed in Figure  7B−G, H2 inhibited neuronal OGD/R damage. The cell survival rate was higher, while the levels of LDH and pro−inflammatory factors in the medium and cell apoptosis rate were Collectively, based on these findings, H2 protects neurons from OGD/R injury by suppressing autophagy.

LincRNA-EPS Knockdown Abolished the Protection of H 2 against Cell Injury
According to the previous results, it was revealed that H 2 upregulated lincRNA-EPS and Sirt1 while inhibiting autophagy under OGD/R injury. However, whether H 2 could inhibit autophagy and upregulate Sirt1 through activating lincRNA-EPS should be clarified. Thus, to examine the function of lincRNA-EPS in the protective ability of H 2 against neuronal OGD/R-induced autophagy and injury, HT22 cells were transfected with siRNA-lincRNA-EPS.
Firstly, the RT-PCR was conducted to examine the knockdown effect of siRNA-lincRNA-EPS after transfection. Figure 7A illustrates that in contrast to the siRNA-NC group, the expression of lincRNA-EPS in the other three siRNA groups was significantly downregulated. Among them, the siRNA2-lincRNA-EPS had the most obvious knockdown effect; therefore, we selected siRNA2-lincRNA-EPS for follow-up experiments. We then detected some indicators to evaluate the neuronal injury. As displayed in Figure 7B-G, H 2 inhibited neuronal OGD/R damage. The cell survival rate was higher, while the levels of LDH and pro-inflammatory factors in the medium and cell apoptosis rate were reduced in the OGD/R+ H 2 group as opposed to those in the OGD/R group. Nevertheless, the protective effects of H 2 were reversed by siRNA-lincRNA-EPS.
3, x FOR PEER REVIEW 12 of 19 reduced in the OGD/R+ H2 group as opposed to those in the OGD/R group. Nevertheless, the protective effects of H2 were reversed by siRNA−lincRNA−EPS.

LincRNA−EPS Mediated the Effects of H2 on Sirt1 Expression and Autophagy
Western blot analysis and RT−PCR were subsequently conducted to determine the

LincRNA-EPS Mediated the Effects of H 2 on Sirt1 Expression and Autophagy
Western blot analysis and RT-PCR were subsequently conducted to determine the levels of Sirt1, Beclin1, and LC3-II expression. Figure 8 shows that H 2 elevated Sirt1 mRNA and protein levels. However, siRNA-lincRNA-EPS combined with H 2 substantially decreased the Sirt1 protein and mRNA levels. Moreover, Beclin1 and LC3-II levels in the OGD/R+ H2 group were elevated, as opposed to those in the OGD/R group, and these variations could be reversed by administering siRNA-lincRNA-EPS.

LincRNA−EPS Mediated the Effects of H2 on Autophagic Flux
Stable transfection of HT22 cells with tandem mCherry−EGFP−LC3B facilitated us to examine the impact of lincRNA−EPS knockdown on autophagic flux in response to OGD/R damage. As illustrated in Figure 9, H2 inhibited autophagy in HT22 cells, which was abolished by siRNA−lincRNA−EPS.

LincRNA-EPS Mediated the Effects of H 2 on Autophagic Flux
Stable transfection of HT22 cells with tandem mCherry-EGFP-LC3B facilitated us to examine the impact of lincRNA-EPS knockdown on autophagic flux in response to OGD/R damage. As illustrated in Figure 9, H 2 inhibited autophagy in HT22 cells, which was abolished by siRNA-lincRNA-EPS.
Collectively, these results indicated that H 2 inhibits autophagy and activates Sirt1 by upregulating lincRNA-EPS under neuronal OGD/R injury.

LincRNA−EPS Mediated the Effects of H2 on Autophagic Flux
Stable transfection of HT22 cells with tandem mCherry−EGFP−LC3B facilitated us to examine the impact of lincRNA−EPS knockdown on autophagic flux in response to OGD/R damage. As illustrated in Figure 9, H2 inhibited autophagy in HT22 cells, which was abolished by siRNA−lincRNA−EPS.

Discussion
It is known that the weight of the human brain makes up only around 2-3% of the body's total mass, but it consumes about 20% of the body's oxygen supply. When an arterial occlusion occurs, it leads to ischemic stroke, and the glucose and oxygen supplied to the ischemic region decrease drastically. In the stroke core, irreversible cell death occurs within minutes due to complete disruption of blood perfusion around the core region (stroke penumbra), with restricted perfusion and impaired function, which may either recover or progress to infarction over time. In addition, the main cerebral functional carrier neurons are relatively lacking in glycolysis and antioxidant enzymes, which determine the high sensitivity and vulnerability of the brain to hypoxia [41,42]. Therefore, hypoxia and reoxygenation constitute an important aspect of the injury mechanism of ischemic stroke and also become the key simulation direction in in vitro models.
Previous studies [35,43,44] have demonstrated that in vitro OGD/R models can largely reflect cerebral ischemic events at the cellular level. Combined with previous studies, OGD generally responds well to the stroke core zone, but it is not believed that neuronal cell ischemia models are located in the penumbra zone. In our study, we used a tri-gas incubator (94% N 2 /5% CO 2 /1% O 2 ) to cause hypoxia in cells and then reoxygenated them for 24 h. Our findings verified that OGD/R causes severe damage to HT22 cells; we examined their morphology and discovered that the cells had become transparent. In addition, their synapses were shortened or even broken, and their adherent capacity had been diminished.
After the successful development of the OGD/R model, the cell survival rate post-OGD/R at different time points was detected by MTT, and it was discovered that the cell survival rate of the HT22 cells after OGD/R treatment gradually decreased with the extension of OGD time and dropped to about 50% of the control group at OGD-6h/R. Combined with the previously reported findings, most of the OGD/R time selected led to a reduction in cell viability to about 50% in the control group [45]. Under our laboratory conditions, to moderate the damage to HT22 cells, we selected OGD-6h/R for follow-up experiments. Subsequently, it was demonstrated that H 2 could improve neuronal survival rate and reduce LDH level, cell apoptosis rate, and levels of pro-inflammatory factors post-OGD/R. We also found that the protective properties of H 2 could be linked to the regulation of the lincRNA-EPS/Sirt1 signaling pathway and the inhibition of autophagic activity. In addition, we confirmed the hypothesis that H 2 could inhibit autophagy by upregulating the lincRNA-EPS/Sirt1 signaling pathway to alleviate OGD/R injury using autophagy inhibitors and activators, as well as transfecting siRNA to knockdown lincRNA-EPS. Consequently, the present study revealed that H 2 plays a protective role against OGD/R damage in HT22 cells by upregulating the lincRNA-EPS/Sirt1 signaling pathway and inhibiting autophagy.
CI/R injury is associated with multiple brain disorders and can be fatal or disabling. CI/R injury can also cause a series of pathological reactions, including the production of oxygen radicals, calcium ion overload within the cell, glutamate-induced neurotoxicity, inflammatory response, cell apoptosis, and autophagy [2,5,6]. Ischemic preconditioning is a potent protective strategy against CI/R injury [46]. Interestingly, many studies have also shown that pharmacological preconditioning (PreCo) or postconditioning (PoCo) is beneficial to the ischemic organ. Subsequently, the main advantage of pharmacological PoCo over ischemic preconditioning or pharmacological PreCo is its added clinical feasibility or clinically applicable [47]. CI/R injury is often seen in unannounced clinical situations, such as cardiac arrest or ischemic stroke. Because CI/R is unforeseeable and inevitable in these situations, PoCo with hydrogen after CI/R appears to be feasible and effective.
Numerous studies have shown that hydrogen possesses antioxidant, anti-inflammatory, anti-allergic, and anti-apoptotic effects, and it can regulate autophagy levels [7,8]. Extensive research has demonstrated the protective function of hydrogen gas or hydrogen-rich saline on a variety of models of organ injury [9][10][11][12]. However, the signaling pathways and molecular mechanisms have remained elusive. Although some research reports indicate that hydrogen's protective properties stem from its ability to stimulate autophagy, others have proven that its beneficial effects originate from its ability to suppress autophagy [48,49]. Our findings illustrated that after OGD/R injury, the cells were severely damaged, which was manifested by the reduced cell survival rate, increased apoptosis rate, elevated levels of LDH and inflammatory factors, and activation of autophagy. After treatment with hydrogen, the cell survival rate increased, and the LDH level, apoptosis rate, levels of pro-inflammatory factors, and autophagic rate were remarkably reduced, demonstrating that H 2 could improve the neuronal OGD/R injury by suppressing autophagy.
Autophagy is an intracellular degradation system that delivers cytoplasmic constituents to the lysosome [28,30]. The Beclin-1 and LC3 were used to detect autophagy in the present study. Beclin-1 has sequence similarity with the yeast Atg6 gene in mammals, which may bind to class III phosphatidylinositol triphosphokinase (Class III PI3K), form a complex, contribute to the formation of autophagosomes, and regulate autophagy; a higher expression level of Beclin-1 indicates a stronger autophagic activity of the cells [50]. LC3 is the signature protein of autophagy, and when autophagy is initiated, cytoplasmic LC3 (LC3-I) changes from cytoplasmic recruitment to autophagosome membrane to LC3-II [51]. Therefore, the occurrence of autophagy can be confirmed by detecting the levels of Beclin-1 and LC3. Our findings illustrated that OGD/R damage can lead to autophagy activation, reduced cell survival rate, increased cell apoptosis rate, and elevated levels of LDH and pro-inflammatory factors. These injuries are significantly worsened when the autophagy is over-activated by autophagic agonists, and the injuries are relieved when autophagic in-hibitors are used. Our study indicated that the neuronal protection of H 2 was abolished by RAPA, in which cell apoptosis rate, LDH level, and levels of pro-inflammatory factors were remarkably elevated, while the cell survival rate was remarkably reduced. The treatment with 3-MA also confirmed the above-mentioned results. Taken together, it was shown that OGD/R activates autophagy and causes severe cell injury, while H 2 can reduce cell injury caused by OGD/R by inhibiting autophagy.
The molecular mechanisms involved in autophagy regulation are very complex and include multiple signaling pathways [27,30]. Histone deacetylase Sirt1 requires NAD+ for its functioning and is implicated in the modulation of numerous biological activities, such as mitochondrial biogenesis, inflammatory response, oxidative stress, energy homeostasis, cell apoptosis, and autophagy in I/R injury. An earlier research report illustrated that hydrogen reduces hepatocyte apoptosis by stimulating the HO-1/Sirt1 pathway in the hepatic I/R injury model [26]. In addition, numerous studies have confirmed that autophagy was inhibited by stimulating the Sirt1 pathway, thus exerting a protective role [27,52]. Multiple investigations have shown that lincRNA-EPS modulates autophagy and has anti-inflammatory and anti-apoptotic properties [21,22,53]. A recent report showed that the exogenous administration of lincRNA-EPS inhibited microglial activation, reduced cytotoxicity and apoptosis, elevated the proliferative rate of neural stem cells in vitro, decreased the permeability of inflammatory cells, and improved neuronal cell death in ischemic regions in animal models of CI/R [54]. In our previous study, we discovered that neuronal lincRNA-EPS gene expression level was elevated following OGD/R, whereas Sirt1 mRNA and protein levels were reduced, and Beclin-1 and LC3II levels were increased; after H 2 was introduced, however, lincRNA-EPS gene expression was further enhanced, Sirt1 mRNA and protein levels were upregulated, and Beclin-1 and LC3II mRNA and protein levels were downregulated. Therefore, we speculate that H 2 has the potential to inhibit autophagy by activating the lincRNA-EPS/Sirt1 signaling pathway and playing a cytoprotective role in OGD/R. To confirm this inference, we used siRNA to knock down the lincRNA-EPS gene and found that after the lincRNA-EPS knockdown, the protective effect of H 2 was partially weakened. Additionally, it was found that Sir1 was downregulated after the knockdown of lincRNA-EPS, and the autophagic activity of cells was also reduced, indicating that H 2 could inhibit autophagy by activating the lincRNA-EPS/Sirt1 signaling pathway and alleviating cell injury resulting from OGD/R.
The current research has several drawbacks. Firstly, it is notable that in vivo tests were not performed. Interestingly, many previous studies have also used cell experiments alone to investigate brain injury [33,43,55]. Therefore, the experiments in vitro can reveal the related mechanism of the neuroprotection of hydrogen at a certain level. We will also expand the model using HT22 cells that go through differentiation conditions in our future research [56]. Secondly, overexpression of lincRNA-EPS was not performed, thus we did not fully confirm that H 2 plays a role in activating the lincRNA-EPS/Sirt1 signaling pathway. Thirdly, we did not assess the regulatory relationship between Sirt1 and autophagy. Last but not least, this study was only a basic experiment, and its clinical effectiveness needs further verification.

Conclusions
In summary, our study highlighted the crucial function of the lincRNA-EPS/Sirt1/autophagydependent pathway in the attenuation impact of H 2 on OGD/R-triggered injury in hippocampal neurons. Our findings further illustrated that lincRNA-EPS could be a possible target for H 2 , and we established a novel experimental foundation for the H 2 therapy of CI/R injury ( Figure 10).
In summary, our study highlighted the crucial function of the lincRNA−EPS/S tophagy−dependent pathway in the attenuation impact of H2 on OGD/R−triggere in hippocampal neurons. Our findings further illustrated that lincRNA−EPS cou possible target for H2, and we established a novel experimental foundation for the apy of CI/R injury ( Figure 10).