Neuroprotective Effects of Dexmedetomidine against Thapsigargin-induced ER-stress via Activity of α 2 Manami Inagaki-adrenoceptors and Imidazoline Receptors

Dexmedetomidine is a potent and highly selective α2-adrenoceptor agonist with sedative, analgesic, and sympatholytic properties, though it also exhibits some affinity for imidazoline binding sites. In addition to its sedative effects, dexmedetomidine exerts neuroprotective effects under ischemic conditions. Invasive incidents such as ischemia or hypoxia induce dysfunctions in energy production or depletion of ATP as well as accumulation and aggregation of abnormal proteins in the endoplasmic reticulum (ER), leading to an ER-stress response. In the present study, we examined whether dexmedetomidine exerts inhibitory effects on apoptosis mediated by thapsigargin-induced ER-stress in SH-SY5Y cells, and proposed a possible underlying mechanism for its neuroprotective effects. We used thapsigargin (TG) to generate an ER-stress response in SH-SY5Y cells. SH-SY5Y cells were pretreated with Dex (1–1000 nM) or receptor antagonists (atipamezole, efaroxan, BU99006, and 2’,5’-dideoxyadenosine) for 1 hour before co-treatment with 1 μM TG for 20 hours. Co-incubation with dexmedetomidine suppressed thapsigargin-induced increases in cytosolic Ca, caspase-4 and -3 activity, eIF2α phosphorylation, and expression of ER-stress biomarkers. Dexmedetomidine treatment also decreased cAMP levels. In the presence of atipamezole or efaroxan, but not BU99006, inhibition of eIF2α phosphorylation and CHOP expression significantly increased following treatment with dexmedetomidine in thapsigargin-treated cells. However, pretreatment with BU99006 enhanced the increase in mitochondrial membrane potential associated with


Introduction
Dexmedetomidine (Dex) is a selective α 2 1 -adrenoceptor agonist widely used to sedate patients under artificial respiration in an intensive care unit, though analgesic effects of the drug have also been observed [ -5]. In addition, research has revealed that Dex exerts neuroprotective effects in nerve tissue under ischemic conditions [6][7][8][9] and exhibits affinity for imidazoline receptors (I 1 -, I 2

10
-receptors) at its imidazole group [ -13]. However, the precise mechanisms underlying these neuroprotective effects remain to be elucidated.
Ischemic and hypoxic conditions often result in widespread cellular damage, including impairment of ER functions [14]. Following transient cerebral ischemia, the ER of nerve cells become swollen and dilated, and polyribosomes disaggregate from the surface of the ER membrane [15].
Such changes are thought to reflect the inhibition of protein synthesis via protein kinase R-like ER kinase (PERK) activation and the arrest of vesicular transport/secretion from the ER due to the unfolded protein response (UPR). Although it is difficult to determine to what extent ER-stress is associated with cellular injuries in the central nervous system following ischemic hypoxia, intracellular morphological abnormalities have been observed in mitochondria as well. Many cases of central neurodegeneration [16] and decreased levels of lymphocytes [17] have been reported following general anesthesia with volatile agents. Such cellular damage may be associated with Ca 2+ SH-SY5Y cells are derived from the thrice-cloned human neuroblastoma cell line (SK-N-SH) and have been widely utilized in studies of various neurodegenerative disorders, including Parkinson's disease, Alzheimer's disease, and traumatic brain injury. Furthermore, research has revealed that long-term exposure to thapsigargin (TG), which is known to induce ER-stress, may induce apoptosis in SH-SY5Y cells [ release from the ER. 18]. Clarifying the mechanisms underlying the inhibitory effects of Dex on ER-stress is critically important in assessing the clinically applicability of such treatment.
Therefore, in the present study, we investigated whether Dex exerts neuroprotective effects and inhibits apoptosis due to ER-stress induced by TG exposure in SH-SY5Y cells, and whether such effects are associated with α 2 -adrenoceptors or I 1 -, I 2

Materials and Methods
-receptors in these cells.  (Figure 1). All treatments were performed under sterile conditions.

Assay of cyclic AMP (cAMP) level
We used the Cyclic AMP ELISA kit (Cayman chemical company, MI, USA) to assess the concentration of Camp in SH-SY5Y cells pretreated with various concentrations of Dex (1-1000 nM).
Both pretreated and non-pretreated SH-SY5Y cells were incubated with their respective α2-adrenoceptor or I1-, I2-receptor inhibitors for 10 minutes, followed which they were treated with TG +Dex 10 nM for 20 hours. Atipamezole is a selective antagonist of α2-adrenoceptors, with an affinity for α2-adrenoceptors 8000 times greater than that for α1-adrenoreceptors. Zhang et al. reported that Dex (50nM) is significantly antagonized by atipamezole (300 nM) [20]. Therefore, we utilized this level of atipamezole (300 nM) in the present study. Efaroxan, on the other hand is an antagonist for both α2-and I1-receptors, and we employed concentrations of 10 µM based on a previous report by Zhang et al [21]. Levels of cyclic AMP were then assessed in accordance with manufacturer's protocol. Ltd., Aichi, Japan) and the respective specific peptide substrates and then incubated at 37˚C for 2 hours. were collected, and protein content was determined by Bio-Rad Protein Assay using bovine serum albumin (BSA) as a standard.

Assay of eIF2α (eukaryotic translation-initiation factor 2α) phosphorylation
We used the eIF2α ELISA kit (Cell Signaling Technology, Inc., MA, USA) to examine the phosphorylation of eIF2α. Following the 20-hour TG +Dex10 nM treatment, cells were fixed and blocked according to manufacturer protocol and subsequently incubated with either anti-phospho-eIF2α (Thr183/Tyr185) or anti-eIF2α (primary antibody), after which they were examined spectrophotometrically at 405 nm with the Spectra Max i3 (Molecular Devices Co., CA, USA).

Assay of C/EBP homologous protein (CHOP)
For quantitative determination of CHOP in SH-SY5Y cell lysates, we used the ELISA kit for DNA damage inducible transcript3 (CHOP) (Uscn Life Science Inc. Wuhan, China). Cells were fixed and blocked as directed in the manual, following which they were examined spectrophotometrically at 450 nm with the Spectra Max i3 (Molecular Devices Co., CA, USA). CHOP levels were then assessed using the Mît-E-ψ™ mitochondrial permeability detection kit (Enzo Life Sciences, Inc., NY, USA).

Change in mitochondrial membrane potential
A collapse in the mitochondrial membrane potential Δψ is one of the earliest indications of apoptosis. We detected changes in mitochondrial Δψ in hepatocytes using fluorescent the mitochondrial probe JC-1 (5,5', 6,6'-tetrachloro-1, 1', 3,3'-tetraethyl-benzimidazolcarbocyanine iodide) [22]. In hepatocytes not undergoing apoptosis, the mitochondrial Δψ remains intact, and the Each measurement was repeated three times. Results are expressed as mean ± standard error of the mean (S.E.M). One-way analyses of variance (ANOVA) were used to compare the effects of various treatments with those of untreated cells. Post hoc testing was performed using Dunnett's test.
Differences with p-values less than 0.05 were considered statistically significant.

Dexmedetomidine decreased intracellular cAMP
Dex binds to the Gi-coupled α2-adrenoceptor, inhibiting adenylyl cyclase activity and downregulating cAMP formation [23]. We investigated whether cAMP is involved in the cytoprotective effects exerted by Dex. Pretreatment with Dex (10, 100 nM) significantly decreased cAMP levels in SH-SY5Y cells exposed to TG-induced ER-stress (Figure 2-A). When cells were incubated with atipamezole or efaroxan prior to co-treatment with Dex (10 nM) and TG (1 μM), cAMP levels significantly increased when compared to TG+Dex 10 treatment alone (Figure 2-B). promoted ER-stress-induced apoptosis. When cells were incubated with atipamezole or efaroxan prior to co-treatment with Dex (10 nM) and TG, caspase-3 activity significantly increased (Figure 3-B).
These data suggest that the inhibitory effect of Dex on ER-stress-induced apoptosis is mediated through the activity of α2-adrenoceptors and I1-receptors.

Figure 4. Effect of dexmedetomidine (Dex) on caspase-4 activity in SH-SY5Y cells.
Caspase-4 activity was measured using the substrate for caspase

Dexmedetomidine prevented TG-induced increase of [Ca2+]i
We next investigated the effect of pretreatment with Dex on [Ca 2+ ]i in TG-induced ER-stress.

Elevated, continuously increasing levels of [Ca 2+
However, when cells were pre-incubated with atipamezole or efaroxan, the TG +Dex-induced As an imidazole derivative, Dex exerts activity on imidazoline receptors as well as α2-adrenoceptors [24]. While some researchers have reported that the cytoprotective effect of Dex is associated with α2-adrenoceptors, others have suggested that these protective effects are associated with the activity of imidazoline receptors. In general, when α2-adrenoceptors are stimulated, Gi proteins are activated, and levels of cAMP decrease [25]. In order to confirm this phenomenon, we nM also showed such decreases). Research has also reported that cAMP levels do not increase with TG treatment alone [26]. Indeed, our results also indicate that TG treatment alone does not induce significant differences in cAMP levels when compared with control treatments. Following pre-treatment with Dex (10 and 100 nM), cAMP levels significantly decreased in TG-treated cells when compared with those observed for TG treatment alone, suggesting that cAMP levels decreased due to the α2-adrenoceptor-stimulating effect of Dex. When cells were treated with atipamezole and/or efaroxan prior to TG +Dex10 Nm treatment, cAMP levels significantly increased, suggesting an inhibitory effect of Dex on cAMP production via α2-adrenoceptor and I1-receptor stimulating actions.
As the detailed affinity of the antagonist efaroxan for α2-adrenoceptors has not yet been clarified, it remains uncertain whether the differences in results for TG +Dex10 nM +atipamezole and TG +Dex 10nM +efaroxan treatment can be attributed to blockage of I1-receptors, though stimulation of I1-receptors does not seem to extensively reduce cAMP production. In addition, though not shown in the figures, treatment with atipamezole or efaroxan alone did not significantly affect cAMP levels.
Without Dex, intrinsic receptor stimulation seems to be very weak, which may explain why no significant differences were observed following inhibition of α2-adrenoceptors.
Researchers have reported that Dex at a concentration of 100 ng/mL (400 nM) induces apoptosis in neutrophils without α2-adrenoceptor stimulation, and that long-term incubation with Dex inhibits peroxide production [27]. Furthermore, studies have reported cytoprotective effects of Dex (1 μM) in the rat hippocampus mediated by increased expression of pERK1/2 rather than α2-adrenoceptor activity [13]. However, other studies have reported conflicting results: Neuroprotective effects of Dex at 3 μg/kg were observed in wild-type mice but not in α2-adrenoceptor-knocked-out mice, suggesting that the response is indeed mediated by the α2-adrenoceptor [28]. However, the levels of Dex used in these reports are not consistent. When Dex was administrated at a loading dose of 0. reports have utilized higher levels of Dex in order to achieve significant results [13,27]. Therefore, we examined the effects of various concentrations of Dex ranging from 1 to 1000 nM on apoptosis mediated by thapsigargin-induced ER-stress.
As indicated in Figure 3 and Figure 4, the activity of caspase-3 as well as ER-stress-induced apoptosis significantly increased following treatment with TG (1µM). When cells were pretreated with Dex (10 nM)-a concentration close to clinical levels-ER stress induced by treatment with TG was suppressed. At 1000 nM, however, the cytotoxic effects of Dex treatment have been observed [30] .
Therefore, we utilized Dex at a concentration of 10nM in the present study. In TG-induced ER stress, Dex suppressed caspase-4 activity, CHOP levels, eIF2α phosphorylation, and ER-stress at the clinically applicable level of 10 nM. These effects of Dex were suppressed by pre-treatment with atipamezole, an α2-adrenoceptor antagonist, and by pre-treatment with efaroxan, an antagonist of both α2-adrenoceptors and I1-receptors. In addition, Dex-induced inhibition of caspase-3 activity was also suppressed by treatment with these antagonists. These results indicate that the inhibitory effects of Dex on the cytotoxic processes induced by ER-stress associated with TG treatment are related to activity at both α2-and I1-receptors. Furthermore, when cells were pretreated with the adenylyl cyclase inhibitor ddAdo, suppression of caspase-3 activity was much greater than that observed in cells treated with TG +Dex10 nM alone. Thus, these results demonstrate that Dex suppresses apoptosis resulting from TG-induced ER stress by decreasing cAMP levels.
Treatment with ddAdo inhibits adenylyl cyclase and suppresses cAMP production. Based on the results of the present study, we speculate that the mechanism by which Dex exerts neuroprotective effects at the clinical concentration of 10 nM involves not only decreases in intracellular Ca ]i levels, leading to mitochondrial disorders. Indeed, the results of the present study revealed that the mitochondrial membrane potential decreased following treatment with TG, and that this decrease was significantly attenuated by a clinically effective level of Dex. However, this effect was not observed for cells pre-treated with atipamezole or efaroxan and was significantly inhibited following pretreatment with BU99006, indicating the additional protective effects of Dex on mitochondria.
2+ via action at α2-and I1-receptors but also protection of the mitochondrial membrane via action at I2-receptors. Clarification of the mechanisms underlying the cytoprotective effects of Dex should help to widen the range of its clinical use and inform further research regarding its applicability in combination therapy for a number of disorders.