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Study on Antidepressant Effect and Mechanism of Crocin Mediated by the mTOR Signaling Pathway

Crocin is a monomer of Chinese traditional herbs extracted from saffron, relieving depression-like behavior. However, its underlying mechanism of action remains unclear. Herein, we explored whether crocin's antidepressant effect depended on the mammalian target of the rapamycin (mTOR) signaling pathway. The model of PC12 cells injury was established by corticosterone, the changes in cell survival rate were tested by the CCK-8 method, and the changes in cellular morphology were observed under a fluorescence microscope. The depression model was established by chronic unpredictable mild stress (CUMS), and its antidepressant effect was estimated by open field test (OFT), forced swimming test (FST), and tail suspension test (TST). Western blot was used to monitor the protein expression. The results showed that crocin could effectively improve cell survival rate and cellular synaptic growth, alleviate the depressive behavior of CUMS mice, and promote the expression of BDNF, P-mTOR, P-ERK, and PSD95. However, when rapamycin was pretreated, the antidepressant effects of crocin were inhibited. In summary, crocin plays a significant antidepressant effect. After pretreatment with rapamycin, the anti-depression effect of crocin was significantly inhibited. It is suggested that the mechanism of the anti-depression effect of crocin may be related to the mTOR signaling pathway. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1007/s11064-022-03668-z.


Introduction
Major depressive disorder (MDD) is a mental disease mainly manifested by depression, pessimism, and functional decline, and its pathogenesis is multiple and persistent. The pathological mechanism of MDD is still unclear, and various causes are often involved. The central nervous system function decline is a significant factor in the onset of MDD. Crocin is a kind of active ingredient extracted from saffron crocus that has a diversiform function in anti-inflammatory, analgesic, neuronal protection, and enhanced memory [1]. In recent years, it has been confirmed that crocin can alleviate neurotoxicity in rotenone-induced Parkinson's disease rats and improve chronic obstructive pulmonary disease-induced depression by inhibiting PI3K/Akt-mediated inflammation [2,3]. Crocin-i can also reduce depressive behavior in mice with chronic corticosterone-induced depression by inhibiting hippocampal neuroinflammation (IL-1β) and oxidative stress and alleviate depression-like behavior by modulating the microbial-gut-brain axis in mice with chronic inhibitory stress [4,5]. Moreover, studies have shown the antidepressant-like effects of crocin by increasing the levels of P-CREB and brain-derived neurotrophic factor (BDNF) and regulating classic antidepressant molecular signaling, like GHSR-PI3K signaling pathway [6,7]. However, the specific mechanism of crocin action was not clearly demonstrated.
Mammalian target of rapamycin (mTOR), a serine/ threonine-protein kinase, and a target of rapamycin and its analogs can be used to regulate cell growth proliferation and protein synthesis [8]. Studies have found that BDNF expression level and activation of the mTOR signaling pathway are essential for rapid-acting antidepressants to promote dendritic growth and increase synaptic protein content [9]. Ketamine, for example, regulated the expression of postsynaptic density protein (PSD95) and Synapsin-1 by activating mTOR and BDNF signaling pathways in mPFC, instantaneously activating downstream 4e binding protein1 (4E-BP1) and p70S6K [10]. In addition, the depressive behavior of CUMS-induced mice is also associated with reduced levels of p-mTOR and p-p70S6K [11]. Consequently, the mTOR signaling pathway was considered crucial for developing new antidepressant drugs. As a specific inhibitor of the mTOR signaling pathway, rapamycin (rapa) has been identified to inhibit the activation of the mTOR signaling pathway, leading to synaptic dysfunction, thereby blocking the effects of rapid-acting antidepressants. What was certain is that crocin had the function of alleviating depressive behavior and regulating the central nervous system [2][3][4], but whether the antidepressant effect of crocin depends on the mTOR signaling pathway was still not clear. Rapamycin, a blocker of mTOR, was used in advance to reveal whether the mechanism of acute antidepressant action of crocin depends on the mTOR signaling pathway at both cellular and animal levels, thus providing evidence for the development of new rapid antidepressants.

Animals
Male ICR mice, 6-8 weeks old, weighing 22 ± 2 g, were obtained from Hunan Slack Jingda Experimental Animal Co., Ltd. SCXK (Xiang) 2019-0004. (Changsha, China). All animals are kept under standard conditions, with a temperature of (23 ± 2) ℃ and relative humidity (50-65%). The light follows the laws of nature, free drinking, and eating. Acclimatize for 7 days before the experiment, and then group according to body weight. Detailed steps are shown in Figs

Delivery Way
Rapamycin and 0.9% NaCl was administered by lateral ventricle injection. Crocin and drinking water were administered by gavage [12].

CUMS Model
CUMS simulates the social pressure of patients with depression. The experimental program adopts orphan rearing and 4 consecutive weeks of stress stimulation except for the control group, including: (1) water deprivation for 24 h, (2) fasting for 24 h, (3) wet litter for 24 h, (4) noise (> 80 dB) for 3 h, (5) tail-clamping for 30 min, (6) tilts the squirrel cage at 45° for 24 h, (7) swim in ice water for 5 min, (8) turn upside down day and night, (9) light up all night [13,14]. Perform 1-2 kinds of stimulation every day to ensure that the stimulation is not repeated within 3 days.

Open Field Test (OFT)
The whole experiment process was carried out in a quiet and dark environment. Before the experiment, the mice were placed in the experimental environment to fit in for 1-2 h. After 30 min of administration, the mice were placed in the bottom center of the square arena (50 × 50 × 50 cm), and the number of horizontal crawls and the number of uprights of the mice within 5 min were recorded. Clean the inner wall and the bottom of the arena to avoid the smell left by the last mice [15].

The Forced Swimming (FST)
The FST is also one of the most commonly used methods to measure the extent of despair in animals. The mice were placed in a beaker (20 × 14 cm in diameter) containing 15 cm deep water at 24 ± 1 ℃ to swim. The cumulative immobility time (s) was recorded for the last 4 min of a 6 min swim test. The mice were supposed to be motionless when they stopped struggling or produced only small limb movements to keep their head floating above water [16].

The Tail Suspension Experiment (TST)
The TST is often used to test the efficacy of preclinical antidepressants. In a soundproof and visually isolated room, the mice were suspended 30 cm from the ground, and the tail of the mice was fixed with tape about 1 cm from the end. Recording the total immobile time of the last 4 min of the 6 min test [16].

Lateral Ventricle Injection Method
The mice were anesthetized with 3% pentobarbital and fixed to a stereotactic brain localization device. When we find the position of the bregma, the anterior fontanelle is the coordinate point, move forward 0.3-0.6 mm, and move laterally 1 mm to locate [17]. Drill a hole with a bone drill, insert a 1.7 mm microsyringe, and inject 2 μL Trypan blue. Wait 1 h after the injection, and open the brain tissue. If the blue diffuses to other ventricles, the location is successful. The buried pipe repeats the above process, and the customized casing and casing cap are embedded in the borehole. Postoperative observation 1-2 days, if there is no discomfort, recovery 5-7 days, and then the experiment. Rapamycin can be injected directly through the casing. The specific steps are shown in Fig. 3B.
After the cells were in the logarithmic growth phase, they were seeded in a 96-well plate with 1 × 10 4 cell mL −1 . After the cells adhered to the wall, different concentrations of CORT (100, 200, 300, 400, and 500 µmol L −1 ) were added, continued to culture for 24 h, then measured the cell survival rate by the CCK-8 method. We selected the CORT concentration at which 50% of the cells were damaged for subsequent experiments. The drug administration sequence was as follows: crocin was pretreated for 2 h, then corticosterone was treated for 24 h, 10 µmol L −1 CCK-8 was added into each well for further culture for 1-4 h, and the OD value was determined at 450 nm with a microplate meter. Cell survival rate (%) = [OD (experimental group) − OD (zeroing group)/OD (control group) − OD (zeroing group)]100%. The synaptic growth was observed and photographed under an inverted fluorescence microscope (× 20).

Western Blot
The prefrontal cortex was lysed in RIPA buffer containing protease inhibitors and phosphatase inhibitors. The protein concentration was quantified by Bradford's method. The protein concentration was measured by BSA, diluted to a uniform concentration, and boiled in boiling water at 100 ℃ for 5 min. The sample was added to sodium dodecyl sulfate-polyacrylamide gel for electrophoresis separation (SDS-PAGE), and then the protein was transferred to the PVDF membrane with a wet transfer buffer and blocked in a 5% skimmed milk powder solution in TBST 1-1.5 h, the membrane was incubated with mTOR (1:1000), p-mTOR (l:1000), BDNF (1:1000), PSD95 (1:1000), ERK (1:1000), P-ERK (1:1000). Detection was performed with an Alpha Fluor Chem gel imager, and data were analyzed using Image J software. Each experiment was performed more than three times [2].

Statistical Analysis
All data were expressed as mean ± SEM and graphed using the GraphPad Prism 8.0.2 (San Diego, USA). IBM SPSS Statistics 26.0 was used for data analysis. One-way analysis of variance (ANOVA) was used when the data conformed to the normal distribution. The non-parametric test was used when the data did not conform to the normal distribution. P < 0.05 is considered statistically significant.

Crocin had a Significant Effect on Protecting the Survival Rate of Damaged Cells and the Growth of Cell Synapses
The results revealed that PC12 cells were damaged in a concentration-dependent manner in response to different concentrations of corticosterone. When the concentration was 500 μmol L −1 , the cell survival rate was about 50% (Fig. 1A; ### P < 0.001), which could be used for subsequent modeling. Before corticosterone was administered, crocin was pretreated for 2 h. The results showed that compared with the model group, all crocin concentrations could significantly improve cell survival rate and promote the growth of cellular synapses (Fig. 1B-D). Among them, 1 μmol L −1 crocin can significantly improve cell survival rate (*P < 0.05) and promote the growth of cellular neurons (**P < 0.01). When the concentration of crocin increased to 10 μmol L −1 and 30 μmol L −1 , the effect was more significant (***P < 0.001).

Effects of Crocin on Behavior and Protein Expression in Normal and CUMS-Induced Mice
Crocin was administered to normal mice by gavage, and the behavior was detected after 30 min of drug administration, as shown in Fig. 2A. The results showed that, compared with the model group, the cumulative immobility time of mice in FST and TST was significantly lower after administration of crocin, and the difference was statistically significant (Fig. 2B, C, ***P < 0.001, *P < 0.05). In CUMS mice (Fig. 2D), the body weight of mice and the number of crossing and rearing of mice in OFT showed no difference On day 0. (Fig. 2E-G). After the 21 days of modeling, compared with the control group, the weight of the CUMS mice was reduced (Fig. 2E, # P < 0.05, ## P < 0.01), and the number of crossing and rearing in OFT were significantly decreased (Fig. 2F, G, # P < 0.05). Chronic treatment with 10 mg kg −1 and 30 mg kg −1 crocin for 14 days could significantly reversed the CUMS-induced reduction in body weight (Fig. 2E, **P < 0.01, *P < 0.05), and the number of crossing and rearing increased significantly in the high-dose crocin group (Fig. 2F, G, **P < 0.01, *P < 0.05). In addition, after the 14th day of administration, we also tested behavioral changes in mice during FST and TST. The results showed that compared with the control group, the accumulated immobility time in FST and TST was significantly increased in the model group (Fig. 2H, I, # P < 0.05). Compared with the model group, the accumulated immobility time of mice in the high-dose crocin group was significantly reduced in FST and TST, and the difference was statistically significant (Fig. 2H, I, **P < 0.01, *P < 0.05).
The above results have proved that continuous administration of crocin could effectively relieve depressive behavior in CUMS mice. In order to further determine the antidepressant pathway of crocin, we detected the expression levels of BDNF, P-mTOR/mTOR, P-ERK/ERK, and synaptic protein PSD95 in the prefrontal cortex of mice in each group. Experimental studies found that compared with normal mice, the expression of BDNF, P-mTOR/mTOR, P-ERK/ERK, and PSD95 in the prefrontal cortex of CUMS mice was significantly inhibited (Fig. 2J-M, ### P < 0.001, # P < 0.05). After OPT open field test; CUMS chronic mild and unpredictable stress. E Body weight. ( # P < 0.05, ## P < 0.01, compared with the control group; **P < 0.01, *P < 0.01, compared with the model group. All comparisons were made within the same time period.). F, G Total acrossing number and rearing number in OFT. ( # P < 0.05, ## P < 0.01, compared with the control group; **P < 0.01, *P < 0.01, compared with the model group. All comparisons were made within the same time period.). H, I Immobility times of CUMS mice in the FST and TST. ( # P < 0.05, compared with the control group; **P < 0.01, *P < 0.05, compared with the model group). J-M Expression of P-mTOR/mTOR, BDNF, P-ERK/ERK and synaptic proteins PSD95. ( # P < 0.05, ### P < 0.001, compared with the control group; *P < 0.05, **P < 0.01, compared with the model group). One-way ANOVA, (Animal experiment, n = 7-8/group; WB, n = 3-4/group) 1 3 14 days of administration, the expression of these proteins increased significantly (Fig. 2J-M, **P < 0.01, *P < 0.05).

mTOR Plays a Major Role in the Rapid-Acting Antidepressant-Like Process of Crocin
In order to further determine the role of mTOR in the rapidacting antidepressant of crocin, we also independently used rapamycin (an antagonist of mTOR) in combination with an effective antidepressant dose of crocin. We gave 0.1 nmol L −1 rapamycin in advance to incubate for 30 min. We then selected the concentration of crocin that was confirmed by the above experiment to significantly improve the cell survival rate (10 μmol L −1 , 30 μmol L −1 ) for co-incubation (Fig. 3A). The results showed that compared with the model group, the cell survival rate of the crocin group was significantly increased (*P < 0.05, ***P < 0.001), but compared with the same concentration of the crocin group, the cell survival rate of the rapamycin pretreatment group was significantly decreased (^P < 0.05). Similarly, rapamycin was administered to the lateral ventricle 30 min in advance in animal models, and the treatment process is shown in the Fig. 3B. The results showed that in the OFT, there was no significant difference in the crossing number and rearing times among each group (Fig. 3C, D). Interestingly, we found that compared with the same concentration of the crocin group, the pretreatment of rapamycin significantly prolonged the cumulative immobility time of mice in FST (Fig. 3E, ^P < 0.05), and also reversed the protective effect of crocin on the behavior of depressed mice. Subsequently, we measured the expression of BDNF, P-mTOR/ mTOR, P-ERK/ERK, and synaptic protein PSD95 in the prefrontal cortex of mice among each group (Fig. 3F-I). We found that crocin increased the expression of these proteins (***P < 0.001, *P < 0.05, **P < 0.01), but this effect could be significantly blocked by rapamycin (^P < 0.05, ^^P < 0.01).

Discussion
Before 2020, depression was already one of the main factors causing the global health burden. The global spread of COVID-19 has further aggravated people's mental health problems and caused the global prevalence of major depression to 27.6% [18]. However, the pathogenesis of depression is still unclear, and existing drugs consist of many problems such as the slow onset of action, many adverse reactions, and significant individual differences [19,20]. Hence, it is urgent to find new and rapid-acting antidepressants. Studies have shown that crocin played a significant effect in relieving symptoms of depression and ameliorating neuronal damage [2][3][4]. PC12 cells were derived from a pheochromocytoma cell line of rat adrenal medulla, and it is often used for in vitro studies of neurological diseases. CORT-treated PC12 cells are also often used to evaluate the effectiveness of antidepressant drugs [21][22][23][24]. We investigated the effects of crocin on the survival rate and neurons of CORT-induced PC12 cells. The results suggested that a single administration of crocin could effectively improve the survival rate of PC12 injured cells and ameliorate the morphology of cellular neurons. Then we established the CUMS model and detected the behavioral changes of mice through OFT, FST, and TST. Our results showed that crocin improved the autonomy and exploratory behavior of mice in a new environment, reduced the cumulative immobility time of FST and TST, and up-regulated the expression level of BDNF, PSD95, P-mTOR, and P-ERK in CUMS mice. It further confirmed that crocin has a significant antidepressant effect. Intraventricular administration can directly pass through the blood-brain barrier and take effect quickly, so it is often used as a method of administration to study the mechanism of action of drugs for mental disorders [25,26]. The intraventricular application of rapamycin quickly blocks the mTOR signaling pathway and inhibits the expression of downstream synaptic proteins. In order to further study the neuroprotective mechanism of crocin, we injected rapamycin into the lateral ventricle to quickly block the mTOR signaling pathway and then gavage crocin 30 mg kg −1 30 min later. Interestingly, we found that after pretreatment with rapamycin, the neurological effects of crocin and the improvement of depression behavior in mice were significantly inhibited, and the expression of proteins such as BDNF was also significantly reduced. In summary, we believe that the rapidacting antidepressant of crocin may be mediated by regulating the mTOR signal.
BDNF is considered one of the most widely distributed neurotrophic factors in the central nervous system. It promotes the survival and differentiation of 5-HT neurons and ^P < 0.05, compared with the crocin group). F-I Expression of P-mTOR/mTOR, BDNF, P-ERK/ERK, and synaptic proteins PSD95. (***P < 0.001, **P < 0.01, *P < 0.05, compared with the model group; ^^P < 0.01, ^P < 0.05, compared with the crocin group). Oneway ANOVA, (Animal experiment, n = 7-8/group; WB, n = 3-4/ group) plays a crucial role in adult brain neurons' function and synaptic plasticity [27,28]. Similarly, BNDF is commonly used to regulate emotions, such as depression and anxiety. Studies have found that the activation of BDNF and its receptor tyrosine kinase receptor B (TrkB) positively stimulated the expression of its downstream signaling pathways, such as mitogen-activated protein kinase (MAPK) and extracellular information-regulated kinase (ERK) [29]. More and more studies were employed to reveal that the treatment of antidepressants can increase the expression level of BDNF in brain tissue and reduce the stress response [30,31]. In this experiment, it was found that the expression level of BDNF in stressed mice and injured cells was significantly reduced but remarkably increased after administration of crocin, and this effect could be blocked by rapamycin. In addition, BDNF is one of the most widely studied factors that have been identified to be capable of activating ERKs in the hippocampus. ERK further activates a series of protein signaling cascades, such as cAMP response elementbinding protein (CREB), which is responsible for regulating neuronal plasticity and memory function [32,33]. Studies have indicated that crocin exerts neuronal protective effects by stimulating the P-CREB-BDNF signaling pathway [6]. Therefore, we have reasons to believe that the ERK signaling pathway may be one of the targets of crocin action.
mTOR is a signaling pathway downstream of ERK, promoting the phosphorylation of eukaryotic initiation factor 4E-BP1 and S6 kinase1 (S6K1) to regulate protein synthesis and further stimulate the regeneration of central nervous system neurons and peripheral nerve axes [8,34]. Studies have identified that ketamine acts quickly by activating the mTOR signaling pathway and then increasing synaptic signaling proteins [35], so mTOR is often regarded as an essential target of rapid antidepressants. As a specific inhibitor of mTOR, rapamycin can bind to the intracellular receptor (FKBP-12) to form a complex to inhibit protein activity [8]. Meanwhile, PSD95, a synaptic protein downstream of mTOR, maintains synaptic plasticity. It plays a crucial role in learning, cerebral ischemia, and oxidative stress. Hence, we hypothesized that crocin targets the BDNF-ERK-mTOR signaling pathway to improve depressive behavior and regulate synaptic protein plasticity. In this study, rapamycin intervention was conducted to investigate the effect of the mTOR signaling pathway on crocin efficacy. Our results also confirmed the hypothesis that a single dose of crocin repaired the damage to cellular neurons, improved depressive behavior, up-regulated the expression levels of BDNF, P-ERK, and P-mTOR, and synaptic protein PSD95 in mice. At the same time, all the above effects were reversed after pretreatment with rapamycin. In conclusion, the antidepressant effect of crocin may be mediated by the mTOR signaling pathway. However, since depression is a complex mental disorder, the antidepressant mechanism of crocin needs to be further determined from various aspects.

Conclusion
As a natural drug component, crocin has rapid-acting antidepressant effects. Its mechanism depends on the activation of mTOR and ERK and the promotion of BDNF and synaptic protein PSD95 expression levels. It provides ideas and a theoretical basis for the clinical development of novel rapid antidepressant drugs.