Dexmedetomidine Regulates Autophagy via the AMPK/mTOR Pathway to Improve SH-SY5Y-APP Cell Damage Induced by High Glucose

Neurodegenerative diseases and postoperative cognitive dysfunction involve the accumulation of β-amyloid peptide (Aβ). High glucose can inhibit autophagy, which facilitates intracellular Aβ clearance. The α2-adrenoreceptor agonist dexmedetomidine (DEX) can provide neuroprotection against several neurological diseases; however, the mechanism remains unclear. This study investigated whether DEX regulated autophagy via the AMPK/mTOR pathway to improve high glucose-induced neurotoxicity in SH-SY5Y/APP695 cells. SH-SY5Y/APP695 cells were cultured with high glucose with/without DEX. To examine the role of autophagy, the autophagy activator rapamycin (RAPA) and autophagy inhibitor 3-methyladenine (3-MA) were used. The selective AMPK inhibitor compound C was used to investigate the involvement of the AMPK pathway. Cell viability and apoptosis were examined by CCK-8 and annexin V-FITC/PI flow cytometric assays, respectively. Autophagy was analyzed by monodansylcadaverine staining of autophagic vacuoles. Autophagy- and apoptosis-related protein expression and the phosphorylation levels of AMPK/mTOR pathway molecules were quantified by western blotting. DEX pretreatment significantly suppressed high glucose-induced neurotoxicity in SH-SY5Y/APP695 cells, as evidenced by the enhanced viability, restoration of cellular morphology, and reduction in apoptotic cells. Furthermore, RAPA had a protective effect similar to that of DEX, but 3-MA eliminated the protective effect of DEX by promoting mTOR activation. Moreover, the AMPK/mTOR pathway was involved in DEX-mediated autophagy. Compound C significantly suppressed autophagy and reversed the protective effect of DEX against high glucose in SH-SY5Y/APP695 cells. Our findings demonstrated that DEX protected SH-SY5Y/APP695 cells against high glucose-induced neurotoxicity by upregulating autophagy through the AMPK/mTOR pathway, suggesting a role of DEX in treating POCD in diabetic patients.


Introduction
Postoperative cognitive dysfunction (POCD) is associated with memory and language problems and might last for months or even years (Xie et al., 2021).Established risk factors for POCD include major (cardiac and orthopedic) surgery and older age (Zhang et al., 2020).The Aβ protein aggregates into amyloid plaques, so the accumulation of Aβ in the brain can lead to neuronal damage, thus worsening POCD (Liu et al., 2016;Shen et al., 2016).Additionally, diabetes mellitus (DM) and hyperglycemia may be associated with POCD and might worsen postoperative neurologic outcomes (Feinkohl et al., 2017).DM is accompanied by micro-and macrovascular changes and neuropathy, all of which may contribute to neurocognitive dysfunction (Alvarez-Almazan et al., 2020).In addition, during major surgery, an increase in plasma glucose is common, with or without the preoperative diagnosis of DM, due to activation of the hypothalamic-pituitary-adrenal axis.Hyperglycemia may last several days after surgery and is a neuropathic factor that potentially contributes to the development of POCD.Thus, both DM and perioperative hyperglycemia may be additional risk factors for POCD (I F, G W & practice P T J C, 2017).Some studies have shown a correlation between neurocognitive dysfunction and hyperglycemia, but the pathological mechanism of hyperglycemia in neurocognitive dysfunction has not been clearly elaborated (Hou et al., 2020a).
As a sedative drug that has a unique mechanism of action, dexmedetomidine (DEX) can prevent delirium and improve long-term cognitive function (Li et al., 2020;Zhang et al., 2019a).DEX exerts a significant protective effect against cerebral ischemia-reperfusion injury in rats (Wang et al., 2017).In addition, DEX attenuates the neurotoxicity of Aβ (Wang et al., 2018a) and has been reported to activate autophagy via the AMPK/mTOR signaling pathway (Yang et al., 2020).Although some studies have shown the neuroprotective effects of DEX and the improvement of DEX for high glucose (HG)-induced neuronal injury (Hou et al., 2020b), its mechanism has not been completely elucidated and is worthy of exploration.
Autophagy plays a significant role in the essential process that maintains cellular homeostasis and stress responses and is an important physiologic process whereby cytoplasmic components, including defective cellular organelles and proteins, are encapsulated by a double-membrane structure and destroyed in lysosomes (Zheng et al., 2021).In mammalian cells, autophagy is inhibited by mTOR and promoted by AMPK, which is also an upstream regulator of mTOR.The suppression of AMPK activity by HG leads to autophagy inhibition (Cai et al., 2018).Hyperglycemia in DM may directly inhibit neuronal cell autophagy, which subsequently enhances Aβ aggregates, further leading to the dysfunction of cells (Miao et al., 2019).
SH-SY5Y-APP cells are a well-established and widely used in vitro model for neural damage caused by Aβ accumulation (Arrozi et al., 2020;Zhang et al., 2020).Based on previously described research, we hypothesized that DEX could protect SH-SY5Y-APP cells against HG-induced damage by upregulating the level of autophagy through the AMPK/mTOR pathway and investigated this target mechanism.
To explore the protective effect of DEX on HG-induced SH-SY5Y/APP cell injury, cells were divided into the control, DEX, HG, and HG + DEX groups.SH-SY5Y/ APP cells were maintained in drug-free and low-glucose medium.Cells in the DEX group were cultured in lowglucose (5.5 mM) medium containing 1 μM DEX for 24 h.SH-SY5Y/APP cells in the HG group were cultured in high glucose (80 mM) for 24 h.Cells were maintained in highglucose (80 mM) medium containing 1 μM DEX for 24 h in the HG + DEX group.
To investigate whether DEX improves SH-SY5Y-APP cell damage in a high-glucose environment by regulating autophagy, cells were divided into the control, HG, HG + DEX, HG + RAPA, HG + DEX + RAPA, HG + 3-MA, and HG + DEX + 3-MA groups.Cells in the HG + RAPA group were maintained in high-glucose medium supplemented with 200 nM RAPA.SH-SY5Y-APP cells in the HG + DEX + RAPA group were maintained in high-glucose medium containing 1 μM DEX and 200 nM RAPA for 24 h.In the HG + 3-MA and HG + DEX + 3-MA groups, SH-SY5Y-APP cells were pretreated with 5 mM 3-MA for 1 h.To explore the pathway by which DEX regulates autophagy, cells were divided into the control, HG, HG + DEX, and HG + DEX + CC groups.Cells in the HG + DEX + CC group were pretreated with 10 mM CC (AMPK inhibitor) for 1 h and then cultured in high-glucose medium containing 1 μM DEX for 24 h.

Assessment of Cell Viability
SH-SY5Y-APP cells were cultured in 96-well plates and differentiated with RA for 5 days.After being exposed to different treatments for 1 day, cell viability was assessed by the Cell Counting Kit-8 (CCK-8, Beyotime Biotechnology, China) assay.Briefly, the culture medium was absorbed and replaced with 10 µl of CCK-8 solution, and the cells were incubated at 37 °C for 1 h.Then, the absorbance was measured at 450 nm optical density with a microplate reader.

Cell Morphology Observation
Cells were seeded in 6-well plates.at a density of 5 × 10 5 cells/well in a final volume of 3 mL/well and incubated at 37 °C for 24 h.Using a phase-contrast light microscope (Leica DMI8, Germany, 20 × objective), morphological changes characteristic of SH-SY5Y-APP cells were observed.Microphotographs in each group were taken randomly with LAS X software.

Flow Cytometric Analysis of Apoptosis
The different groups of cells were stained with FITC-Annexin V and propidium iodide (PI) in the dark with the Annexin V-FITC/PI Apoptosis Detection Kit (BD556547, BD Bioscience, USA) according to the manufacturer's instructions.Then, each sample was washed and analyzed with an Accuri™ C6 flow cytometer (BD Bioscience, USA).

Assessment of Autophagy by Monodansylcadaverine (MDC) Staining
MDC is a fluorescent dye that stains autolysosomes, and it was used to observe cellular autophagy.SH-SY5Y-APP cells were seeded in 12-well plates with cover slips.After incubation with 10 μM MDC (G0170, Solarbio, China) for 45 min in the dark, the cells were washed once with PBS and visualized by fluorescence microscopy (Leica DMI8, Germany, 20 × objective) with an excitation wavelength of 355 nm and emission wavelength of 512 nm.LAS X software was used to acquire images.To measure the mean fluorescence intensity of MDC, Image ProPlus software (Version 6.0, Media Cybernetics, Bethesda, FL, USA) was used for quantitative analysis.

Statistical Analysis
All experiments in this study were repeated at least three times for each condition.Statistical analysis was performed using GraphPad Prism 8.0 software (GraphPad Software Inc. La Jolla, CA, USA).The data were analyzed by oneway analysis of variance (ANOVA) followed by the multiple comparisons test.The data are expressed as the mean ± SD, and the p value threshold was set at ≤ 0.05.

DEX Attenuated High glucose-Induced Injury in SH-SY5Y-APP Cells
SH-SY5Y-APP cells were cultured for different times (12, 24, 48 h) in DMEM containing different concentrations of glucose.Cell viability in each group was examined by CCK-8 assay.The results suggested that HG decreased cell viability in a concentration-and time-dependent manner (Fig. 1a).Based on previous and experimental results (Nie et al., 2018;Salimi et al., 2018), we chose 80 mM high-glucose treatment for 24 h as the optimum treatment condition.For the cells grown in low-glucose medium, no significant difference was observed among the 0, 0.1, 1, 10, and 100 μM DEX-treated groups (Fig. 1b and c).A concentration of 1 μM DEX was used.The changes in cytomorphology and total apoptotic rates in SH-SY5Y-APP cells treated with high glucose were improved by DEX (Fig. 1d-g).

HG Decreased Autophagy in SH-SY5Y-APP Cells
DEX upregulated the level of autophagy in cells injured by HG.Compared with the control group, Beclin-1 protein levels and the LC3II/I ratio were downregulated and p62 protein levels were upregulated in the other groups (Fig. 2a-d).This result indicated that autophagy was inhibited and that autophagic flux was reduced.In HGinduced cells, MDC staining of autophagic vacuoles revealed an increase in fluorescence intensity after exposure to DEX or RAPA (Fig. 2e and f).DEX and RAPA promoted HG-induced SH-SY5Y-APP cell autophagy.3-MA pretreatment decreased autophagic flux in the HG + DEX group, suggesting that as a pharmacological inhibitor of autophagy, 3-MA could suppress DEXinduced autophagy.

Autophagy Affected Cell Activity in SH-SY5Y-APP Cell Injury Induced by HG
Cells in the control group showed good growth and normal morphology.The morphological alterations in HG-induced cells were ameliorated by the administration of DEX and RAPA (Fig. 3a).Flow cytometry with Annexin V-FITC/ PI double labeling was used to measure apoptosis.The results of flow cytometry showed a reduction in the fraction of apoptotic cells in the HG + DEX, HG + RAPA, and HG + DEX + RAPA groups compared with the HG group.With 3-MA pretreatment, the protective effect of DEX was reversed (Fig. 3b and c).

DEX Activated the AMPK/mTOR Pathway in SH-SY5Y-APP Cells Incubated with HG
To verify the association between the pharmacological effects of DEX and the AMPK/mTOR pathway, we examined the expression levels of mTOR, p-mTOR, AMPK, and p-AMPK.There was no significant difference in activation of the AMPK pathway between the control and DEX groups.With HG treatment, the p-AMPK/AMPK ratio was significantly decreased, and the p-mTOR/mTOR ratio was increased.HG suppressed the AMPK/mTOR pathway, and DEX activated the AMPK/mTOR pathway when this pathway was suppressed (Fig. 4).

DEX Upregulated AMPK/mTOR Pathway-Related Protein Expression in SH-SY5Y-APP Cells Cultured in HG and Increased the Level of Autophagy
DEX-induced AMPK negatively regulated mTOR and thereby enhanced autophagic flux.Compared with that in the HG + DEX groups, pretreatment with CC upregulated p62 protein levels and downregulated Beclin-1 protein levels and the LC3II/I ratio (Fig. 5a-d).MDC staining of autophagic vacuoles revealed a decrease in the fluorescence intensity after exposure to CC (Fig. 5e and f).
DEX upregulated autophagy via the AMPK/mTOR pathway so that the damage induced by HG in SH-SY5Y-APP cells.The proportion of apoptotic cells was measured at 24 h after treatment by flow cytometry.Comparing the ratio of apoptotic cells with the HG + DEX group, CC increased both early and late apoptotic cells.Similar results can be seen in cell morphology (Fig. 6d-f).Compared with the HG + DEX group, pretreatment with CC attenuated the upregulation of the protein expression levels of BAX/Bcl-2 and cleaved caspase 3 in the HG + DEX + CC group (Fig. 6a-c).Inhibition of AMPK by pretreatment with CC significantly reversed the protective effects of DEX on SH-SY5Y-APP cells.

Discussion
As an increasingly common metabolic disease, DM is characterized by chronic hyperglycemia, is recognized to lead to cognitive dysfunction and is known to be a risk factor for aggravating Alzheimer's disease (AD) and POCD (Chandrasekaran et al., 2020;Feinkohl et al., 2017;You et al., 2021).This study used high-glucose medium to induce a cell injury model (Nie et al., 2018;Salimi et al., 2018).Our results showed that 50 mM to 100 mM glucose induced SY-SY5Y-APP cell injury and apoptosis after 24 h or 48 h in a concentration-and time-dependent manner.High osmotic pressure may have had secondary impacts on cell viability in culture when the glucose concentration was less than 100 mM (Li et al., 2013).According to these experimental data, 80 mM glucose for 24 h was chosen as the experimental concentration for further experiments.
As a highly selective α 2 -adrenergic receptor agonist, DEX can provide analgesia and sedation without respiratory depression and shows some perioperative neuroprotective activity (Kleiman & Johnson, 2019).There are numerous recent studies on the neuroprotective effect of DEX in different models of nerve injury, including HG-induced models (Hou et al., 2020a;Sun et al., 2020;Wang et al., 2017).However, the specific mechanisms of neuroprotection have not been fully elucidated.The mechanisms may be related to the regulation of autophagy, attenuation of neuroinflammation, and promotion of neurogenesis (Qiu et al., 2020;Zhang et al., 2019b;Zhu et al., 2019).This study confirmed that there was no significant cytotoxicity of DEX at concentrations below 100 µM.At concentrations from 1 μM to 20 μM, DEX had a protective effect against damage in HG-induced SH-SY5Y-APP cells.Based on these data, we determined the optimal concentration of DEX to be 1 μM, as in previous studies (Chen et al., 2019).CCK-8 assays and flow cytometry are commonly used to study the viability and apoptosis of SH-SY5Y-APP cells.The results showed that DEX increased cell viability, improved cell morphology, and reduced cell apoptosis, which was in line with previous results (Hou et al., 2020b).
Aβ deposition in the diabetic brain has been reported (Wakabayashi et al., 2019).Excessive accumulation of Aβ can initiate a pathological cascade that gradually aggravates neuronal dysfunction and eventually manifests as cognitive dysfunction (Zhang et al., 2020).Neural cell death induced by aggregated Aβ plaques affects the pathogenesis of several cognitive dysfunction diseases (Chen et al., 2021).As an in vitro cell model, SH-SY5Y cells are widely used in studying several aspects of neurotoxicity (Wu et al., 2021;Zhang et al., 2021).The SH-SY5Y-APP cell model, which stably overexpresses APP695, has been used to investigate Aβ toxicity (Arrozi et al., 2020;Zhang et al., 2020).The accumulation of Aβ in the brain can lead to neuronal damage, thus worsening AD and POCD (Liu et al., 2016;Shen et al., 2016).Aβ clearance is regulated by autophagy, and upregulation of autophagy can promote Aβ degeneration (Yang et al., 2011).DEX treatment showed protective effects in the SH-SY5Y-APP cell model injured by HG.
Autophagy is one of the major mechanisms by which intracellular Aβ is cleared, so autophagy is related to the accumulation of Aβ, so autophagy could impact the function of SH-SY5Y-APP cells (Kuruva et al., 2017).As one of the most common sedative medications used in the clinic, the effect of DEX on autophagy has gained widespread attention (Oh et al., 2019;Zhao et al., 2020).Our study demonstrated that DEX prevented autophagy inhibition by HG, but this effect was not significant in a low-glucose environment.In SH-SY5Y-APP cells, enhanced autophagy levels may alleviate apoptosis and improve cell morphology.Autophagy is associated with many pathways, including AMPK/mTOR, which is associated with energy metabolism (Cai et al., 2018).mTOR signaling negatively regulates autophagy induction.As an upstream molecule of mTOR, AMPK can inhibit mTOR signaling.The activation of the AMPK/ mTOR signaling pathway contributes to the upregulation of autophagy, and HG culture conditions inhibit AMPK activity (Chen et al., 2018).This study also demonstrated that activation of the AMPK pathway by DEX prevented autophagy inhibition by HG.HG inhibited autophagy through AMPK signaling and resulted in increased apoptosis.DEX activates the AMPK pathway, upregulates the level of autophagy, and downregulates the apoptosis rate in HG-cultured cells.Our study compared the effects of DEX, 3-MA, and RAPA in HG-cultured SH-SY5Y-APP cells (Cheng et al., 2019).The western blotting and MDC results showed the antiapoptotic effect of autophagy.DEX and RAPA increased the level of autophagy in HG-cultured cells, and 3-MA antagonized the effect of DEX.Therefore, the protective effect of DEX was associated with promoting autophagy in HG-cultured SH-SY5Y-APP cells.The AMPK inhibitor CC reversed the protective effect of DEX and visibly increased apoptosis.This idea that DEX activates AMPK is also supported by previous research (Wang et al., 2018b).In models of sepsis and DM, DEX promotes autophagy and plays a protective role.Scarce research is available regarding the protective effect of DEX in an HG environment (Oh et al., 2019;Yang et al., 2020;Zhao et al., 2020).Some scholars have proven the regulatory effect of dexmedetomidine on autophagy in different models (Kho et al., 2021;Yang et al., 2020), but its beneficial effect on neurodegenerative diseases has not been studied.Therefore, we used the cell model for research, and our results will provide inspiration and reference for future research work.
The in vitro HG model can only partially verify the mechanism of diabetic neuropathological changes, which is a limitation of our study.The number of diabetic patients is steadily increasing, reaching 422 million in 2014 (Salunkhe et al., 2018).With the increase in diabetes, the proportion of diabetic patients undergoing surgery also increased.Our research provides a new therapeutic mechanism of POCD and has good potential clinical value.

Fig. 1
Fig. 1 Protective effect of DEX against HG-induced injury in SH-SY5Y/APP cells.a, b Cell viability was examined by CCK-8 assays.c DEX reversed the reduction in cell viability caused by HG. d Changes in cell morphology in different groups.e, f Apoptosis was

Fig. 2
Fig. 2 DEX significantly increased the level of autophagy in HGtreated SH-SY5Y-APP cells.a The LC3II/I ratio and the expression of p62 and Beclin1 in the different groups were determined by western blotting.b-d Densitometric analysis of the western blotting results.e, f MDC staining of autophagic vacuoles and analysis of

Fig. 3
Fig. 3 DEX upregulates autophagy to improve SH-SY5Y/APP cell damage induced by HG. a Changes in cell morphology in different groups.b, c Apoptosis was measured using Annexin V/PI staining

Fig. 6
Fig. 6 DEX upregulated autophagy via the AMPK/mTOR pathway to improve SH-SY5Y-APP cell damage induced by HG. a-c The expression levels of cleaved caspase 3, BAX, and Bcl-2 in the different groups were measured by western blotting.d Changes in cell morphology in different groups.e, f Apoptosis was measured using