Major depressive disorder (MDD) is a prevalent disorder, clinically characterized by persistent low mood, loss of interest, tardiness of thinking and reduced activity (Vance and Winther, 2020). MDD seriously affect the patient’s work life and social inter-human relations. The pathogenesis of depression is complex, and many hypotheses are involved in disorders of monoamine neurotransmitters such as 5-HT, immunity, oxidative stress, and cell apoptosis, inflammatory response (Diniz et al., 2018; Lin et al., 2018). The current treatment mainly includes tricyclic antidepressants, serotonin and norepinephrine reuptake inhibitors, and selective serotonin reuptake inhibitors (Ferrari and Villa, 2017). However, these medicines have limited effects, and only a third of the patients have an improvement in, and more than 30% patients have persistent symptoms (Mahableshwarkar et al., 2015). Therefore, understanding the molecular mechanisms of the pathophysiology of MDD is of great importance for finding better strategies.

Endoplasmic reticulum stress (ERS) is a protective response of cells. A certain degree of ERS can activate the expression of protective molecules such as endoplasmic reticulum molecular chaperones to resist stress and maintain survival. ERS was reported to play a key role in a variety of cell signaling processes such as apoptosis and inflammation (Mao et al., 2020). ERS has received extensive attention in the study of the mechanism of neuronal apoptosis after cerebral ischemia, and its molecular markers include UPR, ATF, PERK, GRP78, and Caspase-12 (Yi et al., 2021). A previous study uncovered that ER stress response pathway is activated in the pathogenesis of depression disorder, suggesting that ER stress stimulation is a new target for the treatment of depression (Nevell et al., 2014). In addition, Oxidative stress (OS) and the decreased function of endogenous antioxidant substances such as superoxide dismutase (SOD) play an important part in the pathogenesis of depression (Galecki et al., 2009). Some scholars have hypothesized that ERS/OS may be a new pathogenetic mode of MDD, and these two kinds of stress and their downstream signaling pathways are expected to be the targets of new therapies (Cao and Kaufman, 2014).

Escitalopram (ESC) is a highly selective serotonin reuptake inhibitor. Previous studies have found that ESC plays a positive neuroprotective role in several ischemic and degenerative central nervous system diseases, which not only improves depressive symptoms, but also has a positive effect on cognitive impairment (Ma et al., 2015). ESC can also improve spatial learning and memory abilities of Alzheimer’s disease patients and depressed rats by promoting synaptic plasticity in the hippocampus and reducing tau phosphorylation level (Montgomery et al., 2001; Ren et al., 2015). We previously found that ESC can inhibit the hippocampal ESR/OS response in chronic unpredictable mild stress-induced rats, reduce the apoptosis of hippocampal cells, and improve the depressive behavior of rats (Yang et al., 2018). However, it remains unclear about the mechanism underlying the regulation of ESC in ESR/OS.

In this study, we investigated the effect of ESC on viability, apoptosis, ERS, and OS of bEnd.3 cells and explored the potential mechanisms by which ESC affects ERS/OS. This study would provide a profound understanding for the mechanism underlying the effect of ESC on ERS/OS.

ERS has been reported to cause cell apoptosis (Lin et al., 2012). In our study, we also found TM-induced ERS triggered apoptosis in bEnd.3 cells. There are several potential mechanisms responsible for the process of ERS-induced apoptosis. First, in this study, we found that PERK-CHOP pathway was activated in in TM-induced bEnd.3 cells, as demonstrated by increased PERK and CHOP protein expression. PERK-CHOP pathway is a main pathway that mediates ERS and drives apoptosis. PERK is an ER transmembrane sensor and exist in the ER lumen. When sustained and severe ERS occurs, the activated PERK induces CHOP expression, which initiates the CHOP-mediated apoptotic pathway, thus leading to apoptosis (Nishitoh, 2012). A previous study discovered that heat-labile enterotoxin could cause intestinal epithelial cell apoptosis via PERK-CHOP pathway (Lu et al., 2017). Another study found that reticulocalbin-1 knockdown activated ERS through the activated PERK-CHOP signaling pathway, thus leading to nasopharyngeal carcinoma cell apoptosis (Huang et al., 2020). Based on our results, we speculated that TM may cause ERS-induced apoptosis via PERK-CHOP pathway. Secondly, in our study, TM-induced bEnd.3 cells showed remarkable increased XBP1 protein expression as compared with the control group. XBP1 pathway has been reported to participate in cell survival, apoptosis, and inflammation (Yap et al., 2020). XBP1 can enter the nucleus and regulate ERS at the transcriptional level, enhance the folding ability of endoplasmic reticulum proteins, thus enhancing cell survival. However, overactive XBP1 pathway may promote apoptosis of vascular endothelial cells and neurons (Zhao et al., 2016). Wang C. et al. (2018) discovered that estrogen receptor antagonist fulvestrant promoted apoptosis of prolactinoma cells through XBP1 pathway. Therefore, XBP1 pathway may promote ERS-induced apoptosis of TM-induced bEnd.3 cells. Finally, ATF6 is a key transcriptional activator during ERS (Niu et al., 2018). The expression of the CHOP protein is under the control of ATF6 during ERS (Yu et al., 2020). ERp29 deficiency was found to have effects on sensitivity to apoptosis through ATF6-CHOP pathway during ERS (Hirsch et al., 2014). Melatonin protects against neuronal apoptosis via suppression of the ATF6-CHOP pathway in a rat model of intracerebral hemorrhage (Xu et al., 2018). The results of this study showed that the expression of ATF6 increased in TM-induce bEnd.3 cells, suggesting that the ATF6-CHOP pathway might be activated.

There is evidence that BBB permeability plays a key role in the development of MDD (Huang and Tan, 2021). MMP9 can damage the tight connections between endothelial cells, degrade the extracavascular basement membrane directly, thus increasing the permeability of BBB (Gorkiewicz et al., 2015). The expression level of serum MMP9 was increased in MDD patients and correlated with MDD severity (Domenici et al., 2010). It was also found that changes in BBB permeability were in accordance with the increased level of MMP9 in traumatic brain injury and stroke (Song et al., 2018). Pgp is an important component of BBB. The expression of Pgp protein in the brain microvascular endothelial cells of MDD patients and depressed rats was down-regulated, which promoted the permeability of BBB to neurotoxic substances (Hawkins et al., 2010). In this study, TM-induced bEnd.3 cells showed significant increased apoptosis rate and decreased survival rate after 24 h, and MMP9 expression was upregulated and Pgp expression was down-regulated, which indicated that this bEnd.3 cell model exhibit dysfunction of high cell permeability. However, ESC remarkably reversed these changes in TM-induced bEnd.3 cells. These results indicated that ESC might inhibit BBB permeability and participate in MDD progress.

eNOS is mainly expressed in vascular endothelial cells and is the main source of endodermal NO. The maintenance of a certain level of NO and eNOS in vivo has a positive protective effect on cells (Luiking et al., 2012). eNOS/NO was reported to exert a vital role in maintain BBB function (Zhang et al., 2019). A previous study reported that when MDD patients develop persistent cerebral hypoperfusion, the oxidative function of mitochondria of endothelial cells can be affected, leading to the increase of ROS synthesis, which in turn leading to the decrease of endothelial eNOS and NO levels, thus weakening the protective effect of them on endothelial cells (Chen et al., 2010). TM reduced the expression and phosphorylation of eNOS and the generation of NO in the rat aortic endothelial cells, which were reversed by ticagrelor (Wang X. et al., 2018). It was reported that TM inhibited eNOS levels, phospho eNOS and NO production in cardiomyocytes, and baicalin inverted these changes and attenuated ERS-induced apoptosis (Shen et al., 2014). Similarly, in the present study, TM-induced ERS/OS resulted in the decreased expression of NO and eNOS in bEnd.3 cells, while ESC reversed these changes, indicating that ESC played a protective role through regulating eNOS/NO. In addition, the Ca²⁺ signaling pathway has been found to play a key role in BBB permeability (Ding et al., 2019). Evidence indicated that after ERS is initiated, endoplasmic reticulum Ca²⁺ leaks into the cytoplasm, and cytoplasmic Ca²⁺ overload activates intracytoplasmic CaMKII, which mobilizes Ca²⁺ to move from ER into the cytoplasm and eventually causes OS (Muneer and Shamsher Khan, 2019). Simultaneously, intracellular Ca²⁺ overload and increased CaMKII expression are necessary for ERS-induced apoptosis (Wang et al., 2017). Similarly, in this study, the Ca²⁺ and CaMKII expression was significantly increased in TM-induced bEnd.3 cells. Therefore, TM may promote BBB permeability of bEnd.3 cells via Ca²⁺/CaMKII pathway.

OS is a main factor for BBB permeability in neurological disorders (Kuriakose et al., 2019). MDA is one of the active products of OS and the change of its concentration can reflect the damage degree of free radicals to the body (Zheng et al., 2018). SOD is the main antioxidant enzyme in the body, which can remove ROS of cells and play a protective role against oxidation (Tabassum and Jeong, 2019). SOD and MDA are often used to evaluate the occurrence of OS and the effect of intervention measures, and SOD is often used to detect the neuroprotective effect of antidepressants (Khan et al., 2011). The results of this experiment showed that MDA concentration was significantly increased and SOD activity was significantly decreased in TM-induced ERS cell model, indicating that ERS-induced OS occurred in this cell model. However, SOD and MDA expression was reversed by ESC + TM treatment, suggesting that ESC can reduce OS of TM-induced bEnd.3 cells. These results indicated that ESC might inhibit BBB permeability via regulating OS, which improving MDD. Additionally, ERS and OS are closely related, and they interact and induce each other (Kunitomi et al., 2020). Studies have suggested that ERS can reduce eNOS activity and gene expression, leading to vascular endothelial dysfunction, suggesting that ERS causes vascular endothelial dysfunction through its induced OS (Lenna et al., 2010; Galán et al., 2014). Wang et al. (2019) discovered that icariin can ameliorate OS of vascular endothelial cells via suppressing ERS. These researches revealed the complex relationship among ERS, OS, and endothelial cell biology. Therefore, ERS/OS is speculated to be an important mechanism leading to changes in neurovascular and BBB permeability. Inhibition of ERS/OS may be a novel strategy for the treatment of diseases associated with BBB hyperpermeability, such as MDD.

Increasing evidences have shown that fluvoxamine, a selective serotonin reuptake inhibitor, promotes neuroprotection and ameliorates depressive behavior by inhibiting ERS-mediated apoptosis (Hosoi et al., 2012; Omi et al., 2014). Our previous animal studies have shown that the improvement of depression behavior in CUMS rats by ESC may be related to the inhibition of ERS/OS (Wan, 2016; Yang et al., 2018). The present study verified some of the results in vitro and found that ESC can reduce the BBB hyperpermeability induced by TM-induced ERS, which was related to the inhibition of ERS/OS. In addition, ESC treatment remarkably reduced the increased protein levels of CHOP, PERK, XBP1, and CaMKII and increased the reduced expression of p-eNOS and NO in TM-induced bEnd.3 cells. Therefore, the potential mechanisms may include inhibition of XBP1, PERK-CHOP pathways, down-regulation of CaMKII expression and inhibition of eNOS decoupling. Combined with the conclusion of previous animal experiments, it can be inferred that ESC may play a positive role in alleviating cognitive dysfunction of MDD and preventing comorbidities via ERS/OS.

In conclusion, TM can induce ERS/OS in bEnd.3 cells, resulting in increased cell permeability. ESC may alleviate TM-induced bEnd.3 cell hyperpermeability disorder through inhibiting the ERS/OS response via XBP1, PERK-CHOP, and CaMKII pathways and decoupling eNOS. This study provides new insights into the potential mechanisms of MDD and offers useful reference value for expanding the use of ESC in the treatment of MDD.

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

YW designed the experiments. LY and ZC prepared the manuscript. LY, ZC, JL, and PD performed the experiments. LY, ZC, and YW analyzed the data. All authors read and approved the final manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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