Dexmedetomidine Alleviates Lipopolysaccharide-Induced Hippocampal Neuronal Apoptosis via Inhibiting the p38 MAPK/c-Myc/CLIC4 Signaling Pathway in Rats

Dexmedetomidine (DEX) has multiple biological effects. Here, we investigated the neuroprotective role and molecular mechanism of DEX against lipopolysaccharide (LPS)-induced hippocampal neuronal apoptosis. Sprague Dawley rats were intraperitoneally injected with LPS (10 mg/kg) and/or DEX (30 µg/kg). We found that DEX improved LPS-induced alterations of hippocampal microstructure (necrosis and neuronal loss in the CA1 and CA3 regions) and ultrastructure (mitochondrial damage). DEX also attenuated LPS-induced inflammation and hippocampal apoptosis by inhibiting the increase of interleukin-1β, interleukin-6, interleukin-18, and tumor necrosis factor-α levels and downregulating the expression of mitochondrial apoptosis pathway-related proteins. Moreover, DEX prevented the LPS-induced activation of the c-Myc/chloride intracellular channel 4 (CLIC4) pathway. DEX inhibited the p38 MAPK pathway, but not JNK and ERK. To further clarify whether DEX alleviated LPS-induced neuronal apoptosis through the p38 MAPK/c-Myc/CLIC4 pathway, we treated PC12 cells with p38 MAPK inhibitor SB203582 (10 µM). DEX had the same effect as SB203582 in reducing the protein and mRNA expression of c-Myc and CLIC4. Furthermore, DEX and SB203582 diminished LPS-induced apoptosis, indicated by decreased Bax and Tom20 fluorescent double-stained cells, reduced annexin V-FITC/PI apoptosis rate, and reduced protein expression levels of Bax, cytochrome C, cleaved caspase-9, and cleaved caspase-3. Taken together, the findings indicate that DEX attenuates LPS-induced hippocampal neuronal apoptosis by regulating the p38 MAPK/c-Myc/CLIC4 signaling pathway. These findings provide new insights into the mechanism of Alzheimer’s disease and depression and may help aid in drug development for these diseases.


Introduction
Alzheimer's disease (AD) and depression are major causes of increasing morbidity and mortality worldwide. According to estimates, approximately 5.4 million people in the USA have AD [1]. Additionally, more than 350 million people worldwide are currently affected by depression [2], which is predicted to be the world's second largest disability-inducing factor by 2030 [3]. Hippocampal injury, including loss of neuronal populations [4], reduced hippocampal volume [5], and massive neuronal death [6], is regarded as a pathological hallmark of AD and depression. Numerous studies have corroborated that apoptosis is implicated in hippocampal injury. Thus, there is a need to investigate potential antiapoptotic drugs to prevent and treat AD and depression.
Lipopolysaccharide (LPS) was found to be abundant in hippocampal lysates of AD brain [7]. LPS can give rise to depression [8]. Li et al. reported that LPS induces hippocampal neuronal apoptosis by activating the c-Myc/ chloride intracellular channel 4 (CLIC4) pathway [9]. The mitogen-activated protein kinase (MAPK) pathway is implicated in hippocampal apoptosis [10]. The MAPK family consists of stress-activated protein kinases (JNK), extracellular signal-regulated kinase (ERK), and p38 MAPK. The JNK signaling pathway has been reported to be involved in hippocampal neuronal apoptosis regulated by oxidative stress [11]. ERK participates in the pathogenesis of learning disabilities in children by activating hippocampal apoptosis [12]. In addition, p38 MAPK has been demonstrated to mediate apoptosis via multiple mechanisms, including increased c-Myc expression [13], Bax transposition, and caspase-3 activation [14]. However, whether the MAPK pathway mediates LPS-induced hippocampal apoptosis is unknown. Moreover, which member of the MAPK family is involved in the process of LPSinduced hippocampal apoptosis through regulation of the c-Myc/CLIC4 pathway is unknown.
Dexmedetomidine (DEX) is a neurological drug with multiple biological effects including anti-inflammation [15], anti-oxidative stress [16], and anti-apoptosis [17]. Recently, it has been reported that DEX exerts neuroprotective effects in various brain injury models [18,19]. However, the specific underlying molecular mechanism is complex and not well understood. Importantly, DEX can reverse LPS-induced neuronal apoptosis [20]. Additionally, DEX has been shown to weaken apoptosis by inhibiting MAPK pathway activation [21]. DEX can also alleviate apoptosis through regulating the p38 MAPK/ ERK pathway and ROS/JNK pathway [22,23]. However, the detailed molecular mechanism of the protective effect of DEX on LPS-induced hippocampal apoptosis remains unclear.
In the present study, we investigated the effects of DEX on the MAPK family and c-Myc/CLIC4 pathway in LPSinduced hippocampal apoptosis in vivo. We also investigated whether the p38 MAPK/c-Myc/CLIC4 pathway is involved in the protective mechanism of DEX against LPS-induced hippocampal apoptosis by using the p38 MAPK inhibitor (SB203582) in vitro.

Animals and Treatments
Forty-eight male Sprague Dawley rats (6 weeks old, 190-210 g) were provided by the Central Hospital of Harbin Medical University (Harbin, China). Rats were maintained under standard conditions and were given ad libitum access to water and standard rodent pellet food [24]. All animal procedures were conducted according to the Animal Ethics Committee of the Northeast Agricultural University (SRM-11, China).
After a week of acclimatization, rats were divided at random into four groups (n = 12 per group): CON, LPS, LPS + DEX, and DEX. The LPS group was given LPS (Escherichia coli 0111: B4; Sigma-Aldrich, San Francisco, USA) dissolved in 0.9% physiological saline at 10 mg/kg by intraperitoneally (i.p.) injection. The LPS + DEX group was administered DEX (30 µg/kg, i.p.; Wuhan Belka Biomedical Co., Ltd, Wuhan, China) 0.5 h before LPS (10 mg/ kg) administration. The DEX group was treated with DEX (30 µg/kg, i.p.). CON group rats were administered with an equal volume of physiological saline by i.p. injection. The doses of DEX and LPS were chosen based on previous reports [9,25,26].

Sample Collection
Four hours after the final treatment, all rats were anesthetized with isoflurane (Yipin Pharmaceutical Co., Ltd, Hebei, China) and sacrificed. Blood was quickly collected by cardiac puncture and centrifuged at 3000 rpm for 10 min at 4 °C. The supernatant was collected for inspection of inflammatory indicators. The whole brains of three rats in each group were immediately fixed in 10% formalin solution for histopathological observation and immunohistochemical detection. Three hippocampal tissues per group were used for ultrastructural observation. The remaining six hippocampal tissues in each group were rapidly frozen in liquid nitrogen and then stored at − 80 °C for real-time PCR and western blot analysis.

Histopathology and Ultrastructural Observation
Brain tissue samples fixed with 10% formalin for 24 h were dehydrated and then embedded in paraffin. Next, they were cut into 4-5-µm sections and stained with hematoxylin and eosin (H&E; Wuhan Biotechnology Ltd. Co., Wuhan, China). After 5-10 min, all sections were observed and photographed under an optical microscope (TE2000, Nikon, Japan). The number of neurons in hippocampal CA1 and CA3 regions was counted using ImageJ software (400 × magnification).
After fixation with 3% glutaraldehyde for 48 h, the hippocampus blocks (1 mm 3 ) were exposed to 1% osmium tetroxide for 2 h. Next, the blocks were dehydrated, embedded, sectioned (60 nm), and then stained with lead citrate. All samples were captured by transmission electron microscope (Tecnai-G212, FEI Company, Netherlands). The mitochondria in six discontinuous fields of each sample were scored based on the morphology and integrity of the mitochondrial membrane and cristae. The criteria for scoring mitochondrial damage were as follows: 0, well-defined and organized membranes and cristae; 1, minor distortions and/or swellings, but general organization retained; 2, major distortions and/or swellings and discontinuous membranes and cristae; 3, membranes and cristae dissociated into particulates to produce diffuse mitochondrial ghosts; 4, only a few mitochondrial remnants in cells.

Immunohistochemistry Analysis
Immunohistochemical analysis was performed as described previously [27]. Briefly, paraffin-embedded brain tissue slices were dewaxed with xylene, dehydrated with gradient alcohol, incubated with hydrogen peroxide, and sealed with goat serum. After that, the sections were incubated with primary and secondary antibodies and labeled with horseradish enzyme. DAB was used for color development. Finally, all slices were observed and photographed under a DP73 type microscope (OLYMPUS, Japan).

Cell Culture and Drug Treatments
PC12 cells were obtained from the Chinese Academy of Sciences (Shanghai, China) and cultured in DMEM medium (Gibco, Waltham, MA, USA) containing 10% FBS and 1% penicillin-streptomycin in a humidified incubator (37 °C, 95% relative humidity, 5% CO2). The experimental groups were as follows: CON, LPS, LPS + DEX, and LPS + SB. CON group cells were cultured in untreated medium. LPS group cells were cultured in medium supplemented with LPS. LPS + DEX group cells were treated with DEX for 30 min and then cultured in medium supplemented with LPS. LPS + SB group cells were administered with p38 MAPK inhibitor SB203582 (10 µM, Selleck.cn, Shanghai, China) for 30 min and subsequently cultured in medium with LPS. The concentrations of LPS and DEX were determined by CCK-8 assay.

Cell Viability Assay and Observation of Cell Morphology
Cells were seeded into 96-well plates (4 × 10 3 cells per well) and cultured in a humidified incubator for 12 h. Next, the cells were treated with DEX and LPS as described. The cells were then allowed to continue cultivating in a humidified incubator at 37 °C and 5% CO2. After 24 h of incubation, the cell viability was determined with a CCK-8 kit (Beyotime Institute of Biotechnology, Suzhou, China) according to the manufacturer's instructions. The absorbance was read at 450 nm by a Bio-Tek Epoch microplate reader (Bio-Tek, Winooski, VT, USA). Cell morphology of each group was observed and captured by inverted microscope (Leica, Germany).

LDH and ATP Release Assay
Released LDH, ATP content, and total ATPase activity were determined by LDH assay kit, ATP content assay kit, and ultra-micro total ATPase kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), respectively.

Dual Immunofluorescence and Annexin V-FITC/PI Staining Assay
Cells in each group were fixed with 4% paraformaldehyde for 30 min and were then permeated with 0.3% Triton X-100 for 10 min. Next, they were incubated overnight with Bax (Wanlei, Shenyang, China) at 4 °C. The cells slides were incubated with CY3-labeled anti-rabbit IgG (Goodbio Technology, Wuhan, China) in the dark for 1.5 h at 37 °C, followed by washing three times with phosphate buffered saline. Afterwards, the cells were incubated overnight with Tom20 (ABclonal, Wuhan, China) at 4 °C. Finally, they were incubated with 488-anti-rabbit IgG (Goodbio Technology) in the dark for 1.5 h at 37 °C. Images were captured using a fluorescent inverted microscope (Nikon).
The resuspended cells were incubated with 5 µL Annexin V-FITC and 10 µL propidium iodide (Bioss, Beijing, China) for 15 min. Then, cell apoptosis was detected by the Attune NxT flow cytometer (Thermo Fisher Scientific).

Real-time PCR Analysis
Total RNA was isolated from hippocampal tissue and PC12 cells by using Total RNA Extraction Kit (Promega Biotech Co, Ltd, Beijing, China). Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA) was used for reverse transcription to obtain cDNA according to the manufacturer instructions. Primer sequences of c-Myc, CLIC4, and GAPDH are shown in Table 1. RT-PCR analysis was performed using LightCy-cler480 (Roche, Basel, Switzerland). The relative quantification of the target gene expression was calculated according to 2 −ΔΔCt method.

Western Blot Analysis
Hippocampal tissue and PC12 cells were lysed in RIPA buffer with phenylmethanesulfonyl fluoride and phosphatase inhibitor (Beyotime Biotechnology, Shanghai, China). After the protein concentration was determined using the BCA assay kit, the equivalent amount of protein samples was separated by SDS-PAGE gel electrophoresis, and transferred to the PVDF membranes. Then, membranes were blocked in 5% skim milk for 2 h at room temperature. Subsequently, membranes were incubated with primary antibodies in Primary Antibody Dilution Buffer (Leagene Biotechnology, Beijing, China) overnight at 4 °C. The primary antibodies included Bax, cleaved caspase-3, and cleaved caspase-9 (1:1000, Cell Signaling Technology, USA); Bcl-2, cytochrome C, P-JNK, JNK, P-ERK, ERK, P-p38, and p38 (1:750, Wanlei); and c-Myc, and CLIC4 (1:1000, Santa Cruz Biotechnology Inc., USA). Next, the membranes were incubated with the appropriate combination of secondary antibody for 2 h at 37 °C. The immunoreactive protein bands were visualized using the enhanced chemiluminescence kit (Beyotime Biotechnology), and were captured by Amersham Imager 600 software (GE, USA). Finally, all protein bands were quantified with ImageJ software.

Statistical Analysis
All data were represented as mean ± SD and analyzed using IBM SPASS Statistics 23 software (SPASS, IL, USA). Statistical analysis was conducted via one-way ANOVA, followed by Tukey's post hoc test. Values with P < 0.05 were considered statistically significant, and P < 0.01 were considered extremely significant.

DEX Improved LPS-Induced Hippocampal Structural Lesion
H&E staining revealed histopathological changes of the hippocampus ( Fig. 1a and b). In the CON and DEX groups, neurons in hippocampal CA1 and CA3 regions were arranged neatly and tightly, with round or oval nuclei and clearly visible nucleoli. In the LPS group, the neuronal arrangement was disordered, the intercellular space was increased, and nuclear pyknosis, nucleolus disappearance, partial cell lysis, and a large number of apoptotic and necrotic cells were observed. However, LPS-induced microstructural damage in hippocampal CA1 and CA3 regions was markedly ameliorated by DEX. Moreover, DEX significantly reversed the LPS-induced decrease in the number of neurons in hippocampal CA1 and CA3 regions (P < 0.01, Fig. 1c and d).
Ultrastructural observation showed that no abnormalities were present in the mitochondria, nuclear membrane, and other organelles of hippocampal neurons in the CON and DEX groups (Fig. 1f). In contrast, most of the mitochondria in the LPS group were severely distorted and swollen, mitochondrial membranes were discontinuous, mitochondrial cristae were dissolved and disappeared, and mitochondrial ghosts were occasionally seen. However, DEX treatment visibly reversed the LPS-induced mitochondrial damage (P < 0.01, Fig. 1e).

Effects of DEX on Proinflammatory Cytokine Levels
As shown in Fig. 2, the serum levels of IL-1β, IL-6, IL-18, and TNF-α in the LPS group were significantly higher than those in the CON group (P < 0.01). DEX treatment effectively suppressed the LPS-induced increase of IL-1β, IL-6, IL-18, and TNF-α. Changes in

DEX Attenuated LPS-Induced Hippocampal Apoptosis via Mitochondrial Pathways
As shown in Fig. 3, the protein expression levels of Bax, Bax/Bcl-2, cytochrome C, cleaved caspase-9, and cleaved caspase-3 in the LPS group were markedly elevated compared with those in the CON and DEX groups (P < 0.01). DEX effectively suppressed the upregulation of mitochondrial apoptosis-related protein expression (P < 0.01).
Cell morphology observations showed that the CON group cells were spindle-shaped or polygonal (Fig. 5c). In contrast, cells in the LPS group were round and the number of cells was noticeably reduced. After treatment with DEX and SB203582 (p38 MAPK inhibitor), the number of cells increased, most of them were fusiform or polygonal, and a few were round.
In the LPS group, LDH level was significantly increased, and ATP content and total ATPase activity were markedly decreased compared with those in the CON group (P < 0.01, Fig. 5d-f). However, DEX and SB203582 dramatically reversed all of these changes induced by LPS (P < 0.01).

DEX Suppressed LPS-Induced Activation of the p38 MAPK/c-Myc/CLIC4 Pathway in PC12 Cells
The expressions of P-p38, c-Myc, and CLIC4 were markedly elevated by incubation with LPS. Pretreatment with DEX and SB203582 strongly blocked these increases (P < 0.01, Fig. 6).

DEX Ameliorated LPS-Induced Apoptosis in PC12 Cells
To investigate whether DEX attenuates LPS-induced apoptosis by regulating the p38 MAPK/c-Myc/CLIC4 signaling pathway, Bax and mitochondria co-localize, annexin V-FITC/ PI staining assay, and mitochondrial apoptosis pathwayrelated protein expression detection were performed. As shown in Fig. 7, there were more Bax and Tom20-positive cells in the LPS group than in the CON group. In contrast, these positive cells were noticeably reduced in the LPS + DEX and LPS + SB groups compared with those in the LPS group.
Annexin V-FITC/PI staining assay was used to evaluate PC12 cell apoptosis. Incubation with LPS significantly increased the rate of apoptosis compared with that of the CON Fig. 2 Effects of DEX on proinflammatory cytokine levels. Serum levels of (a) IL-1β, (b) IL-6, (c) IL-18, and (d) TNF-α. Data were presented as mean ± SD (n = 3). ** P < 0.01 vs CON group. ## P < 0.01 vs LPS group group, and the increase was diminished in the LPS + DEX and LPS + SB groups (P < 0.01, Fig. 8a and b). The protein expressions of Bax, Bax/Bcl-2, cytochrome C, cleaved caspase-9, and cleaved caspase-3 were significantly increased by incubation with LPS, whereas pretreatment with DEX and SB203582 prevented these alterations (P < 0.01, Fig. 8c-i). Data were presented as mean ± SD (n = 3). ** P < 0.01 vs CON group. ## P < 0.01 vs LPS group Discussion LPS-induced animal models are widely used to study AD and depression. AD and depression are both characterized by hippocampal dysfunction [28]. The clinical symptoms of AD include memory disturbance, behavioral derangement, and cognitive impairment [29]. Late-life depression has been reported to be related to cognitive impairment [30]. The hippocampus, especially the CA1 and CA3 regions, represents a vital neural control center for behavior, emotion, learning Fig. 4 Effects of DEX on the MAPK/c-Myc/CLIC4 signaling pathway. a Protein expression of P-JNK, JNK, P-ERK, ERK P-p38, p38, c-Myc, and CLIC4 in hippocampus. Protein quantitative analysis of (b) P-JNK, (c) JNK, (d) P-JNK/JNK, (e) P-ERK, (f) ERK, (g) P-ERK/ ERK, (h) P-p38, (i) p38, (j) P-p38/p38, (k) c-Myc, and (l) CLIC4. m c-Myc and n CLIC4 mRNA expression analysis. o Immunohistochemistry of P-p38 protein in the hippocampus (magnification 200 × , scale bars = 50 µm). Data were presented as mean ± SD (n = 3). * P < 0.05, ** P < 0.01 vs CON group. # P < 0.05, ## P < 0.01 vs LPS group and memory, and cognition [31]. Alterations in hippocampal structure are implicated in cognitive decline [32]. In the current study, DEX ameliorated the LPS-induced hippocampal microstructural and ultrastructural lesions, which presented as decreased necrotic neurons, increased neuronal density in CA1 and CA3 regions and improved mitochondrial damage. The findings demonstrated that DEX reversed pathology associated with AD and depression. Thus, DEX may be a promising drug for the prevention and treatment of AD and depression.
Neuroinflammation has been reported to be involved in the occurrence and development of neurodegenerative disorders (such as AD) and is considered to be an important driving factor for cognitive impairment [33]. Numerous studies have demonstrated that systemic administration of LPS can trigger neuroinflammation in the brain [34,35]. LPS binding to Toll-like receptor 4 activates multiple signaling pathways and ultimately leads to increased transcription of proinflammatory cytokines TNF-α, IL-1β, IL-6, and IL-18. In the present study, DEX suppressed the LPS-induced increase of the proinflammatory factors TNF-α, IL-1β, IL-6, and IL-18, which provides further evidence for the neuroprotective effect of DEX.
Apoptosis is implicated in the inflammatory response. However, most reports of LPS models of AD and depression have focused on neuroinflammation. Therefore, the understanding of the pathogenesis of AD and depression is limited, which hinders the development of effective therapeutic c Cell morphology observation in the CON, LPS, LPS + DEX, and LPS + SB groups, at 200 × magnification, bars = 50 µm. d LDH activity. e ATP content. f Total ATPase activity. Data were presented as mean ± SD (n = 6). ** P < 0.01 vs CON group. ## P < 0.01 vs LPS group drugs. Our previous studies have demonstrated that apoptosis is involved in the pathological process of LPS-induced neurodegenerative diseases [9]. Nanhui Yu et al. reported that inhibition of neuronal apoptosis alleviated cognitive impairment in AD model mice [36]. In addition, reducing apoptosis improved depression-like behavior in rats [37]. The present results demonstrated that DEX attenuated hippocampal neuronal apoptosis induced by LPS, which may be a potential neuroprotective mechanism of DEX against AD and depression.
The MAPK pathway is a major signal transduction pathway that regulates apoptosis [38]. Perturbations in MAPK signaling contribute many human diseases, including AD [39]. The MAPK family consists of JNK, ERK, and p38. It has been reported that JNK is an important potential therapeutic target for depression [40]. Suppression of JNK Fig. 6 DEX suppressed LPSinduced activation of the p38 MAPK/c-Myc/CLIC4 pathway in PC12 Cells. a Protein expression of P-p38, p38, c-Myc, and CLIC4. Protein quantitative analysis of (b) P-p38, (c) p38, (d) P-p38/p38, (e) c-Myc, and (f) CLIC4. g c-Myc and h CLIC4 mRNA expression analysis. Data were presented as mean ± SD (n = 3). ** P < 0.01 vs CON group. ## P < 0.01 vs LPS group attenuated hippocampal neuronal apoptosis and improved depression-like behavior in mice [41]. In addition, dephosphorylation of ERK leads to neuronal apoptosis, which contributes to depression [42]. Recent studies have shown that LPS can promote the phosphorylation of JNK and ERK in a variety of in vitro and in vivo models [43,44]. Surprisingly, our results showed no significant increase in the protein expressions of P-JNK, JNK, P-ERK, and ERK in the hippocampus of rats treated with LPS. The inconsistency with previous studies may result from different models, different injury sites, different mechanisms MAPK pathway activation, and different methods of LPS administration. The p38 signaling pathway can be activated by various signals including LPS [45]. The activated p38 MAPK cascade can induce neuronal apoptosis and lead to hippocampal neuronal damage [46]. Consistent with previous reports, in the present study, phosphorylated p38 was activated and involved in the process of LPS-induced neuronal apoptosis. However, non-phosphorylated p38 did not change significantly. Notably, DEX pretreatment markedly inhibited the phosphorylation of p38. These results suggest that inhibition of p38 phosphorylation may be a Fig. 7 DEX inhibited the translocation of apoptotic protein Bax to mitochondria. The co-localization of Bax (red) and mitochondria (Tom20, green) was measured by double-labeled immunofluores-cence assay (n = 3). Merge is the combination of Bax, Tom20, and DAPI (nucleus, blue). All microphotographs were observed and captured at 400 × magnification, bars = 20 µm protective mechanism for DEX to improve LPS-induced hippocampal apoptosis.
Furthermore, we found that DEX inhibited the LPSinduced increase in the protein and mRNA expressions of c-Myc and CLIC4 in the rat hippocampus. Recent studies have reported that p38 promotes c-Myc activation [47]. c-Myc, an effective apoptosis inducer, binds to the CLIC4 promoter and contributes to the activation of its transcription [48]. CLIC4 is a key mediator of c-Myc-induced apoptosis. Inhibition of CLIC4 activation suppresses c-Myc-induced apoptosis under various stress conditions. Notably, the c-Myc/CLIC4 signaling pathway is involved in LPS-induced Annexin-V/PI positive rate. c Protein expression of Bax, Bcl-2, cytochrome C, cleaved caspase-9, and cleaved caspase-3. Protein quantitative analysis of (d) Bax, (e) Bcl-2, (f) Bax/Bcl-2, (g) cytochrome C, (h) cleaved caspase-9, and (i) cleaved caspase-3. Data were presented as mean ± SD (n = 3). ** P < 0.01 vs CON group. ## P < 0.01 vs LPS group apoptosis of hippocampal neurons. Therefore, we hypothesized that DEX exerts neuroprotective and anti-apoptotic effects by regulating the p38/c-Myc/CLIC4 signaling pathway. This hypothesis was confirmed by observing the effect of the p38 inhibitor SB203582 in LPS-treated PC12 cells. Like DEX, SB203582 significantly reduced the protein and mRNA expressions of c-Myc and CLIC4. Moreover, as assessed by flow cytometry, SB203582 protected PC12 cells from LPS-induced apoptosis. Overall, our findings revealed that the p38 MAPK/c-Myc/CLIC4 signaling pathway was involved in the neuroprotective mechanism of DEX against LPS-induced apoptosis.
CLIC4 is enriched in the mitochondrial outer membrane and is implicated in mitochondrial pathway apoptosis. Overexpression of CLIC4 decreased mitochondrial membrane potential, causing cytochrome C to be released into the cytoplasm, thereby activating caspase [49]. Studies have shown that CLIC4 and Bax synergistically induce apoptosis, but their direct physical interaction have not been detected [50]. Bax, a pro-apoptotic protein, is present in the cytoplasm as a monomer in an inactive state. Under stress conditions, Bax is activated and translocated to mitochondria, forming a heterodimer with Bcl-2, leading to the release of cytochrome C. Thus, Bax/Bcl-2 is regarded as a switch that triggers apoptosis [51]. Our results showed that DEX and SB203582 blocked the translocation of Bax to mitochondria and downregulated the protein expressions of Bax/Bcl-2, cytochrome C, cleaved caspase-9, and cleaved caspase-3. Collectively, our findings suggest that DEX inhibited LPSinduced apoptosis in the mitochondrial pathway by regulating p38 MAPK/c-Myc/CLIC4.

Conclusion
In summary, our results demonstrated that DEX inhibited the LPS-induced activation of the p38 MAPK/c-Myc/CLIC4 signaling pathway and downregulated the expression of mitochondrial apoptosis pathway-related proteins, thereby reducing neuronal apoptosis. These findings provide insight on the neuroprotective mechanism of DEX in LPS-induced hippocampal injury from the perspective of anti-apoptosis, which provides support for the clinical application of DEX in the treatment of AD and depression.