Application of 4-isopropyltoluene to improvement of depression-like behaviors of mice

Abstract

The invention discloses application of 4-isopropyltoluene to improvement of depression-like behaviors of mice. An experiment adopts a chronic constraint stress method to construct a depression model, through behavioral detection such as a sucrose preference test, a tail suspension test and a novel inhibition ingestion test, and in combination with molecular biology means such as immunofluorescent staining and Western blot, the anti-depression effect and potential mechanism of 4-isopropyltoluene are systematically evaluated, and the anti-depression effect and potential mechanism of 4-isopropyltoluene are evaluated. Results show that 4-isopropyltoluene plays a significant anti-depression role by inhibiting neuroinflammation, enhancing glial cell support function and activating AKT signal pathway, and has the potential of being developed into a novel anti-depression candidate drug.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to application of 4-isopropyltoluene in improving depression-like behavior of mice. BACKGROUND

[0002] Depression is a chronic mental disorder characterized by persistent low mood and impaired cognitive function, and its global incidence is increasing year by year. However, the existing first-line clinical antidepressants (SSRIs, SNRIs, NaSSA, etc.) generally have a delayed onset (2-4 weeks), a low clinical remission rate (about 30%), significant side effects (sexual dysfunction, weight gain, metabolic disorders), and about 30% of patients have no response to treatment. Therefore, finding antidepressant candidates with faster onset, higher safety and novel mechanisms has become an urgent need for the development of drugs for mental diseases.

[0003] 4-Isopropyltoluene is a natural monoterpene aromatic compound, which is widely present in cumin, cumin, eucalyptus and various citrus plant essential oils, and has been recognized as GRAS (Generally Recognized As Safe) by FDA as a flavor and food additive. Previous studies have mainly focused on its antioxidant, antibacterial, anti-inflammatory, analgesic and antitumor activities. In recent years, researchers have found that some monoterpenes (such as limonene, linalool) can pass through the blood-brain barrier and regulate monoaminergic, GABAergic and glutamatergic neurotransmission, thereby exerting rapid antidepressant effects, suggesting that 4-isopropyltoluene with similar structure may have yet to be revealed central activity. However, as of now, there is no public literature or patent reporting the direct effect of 4-isopropyltoluene on depression-like behavior, and its neuropharmacological mechanism is also blank. SUMMARY

[0004] The purpose of the present application is to provide the application of 4-isopropyltoluene or its pharmaceutically acceptable salt in the preparation of antidepressants.

[0005] The application adopts chronic restraint stress method (CRS) to construct a depression model, and through sucrose preference test, tail suspension test, novelty suppressed feeding test and other behavior detection, combined with immunofluorescence staining and Western blot and other molecular biology means, the anti-depression effect of 4-isopropyl toluene and its potential mechanism are evaluated. The results show that: (1) on the behavior level: 4-isopropyl toluene significantly improves the sucrose preference rate of the model mice, restores the hedonic deficit; shortens the immobility time in the tail suspension test, enhances the motivation of survival and exploration; reduces the feeding latency of the novelty suppressed feeding test, and relieves the anxiety-like behavior. (2) endocrine level: 4-isopropyl toluene significantly reduces the concentration of peripheral blood corticosterone, suggesting that it can reduce the overactivation of hypothalamus-pituitary-adrenal (HPA) axis. (3) cell and molecular mechanism: promotes the proliferation and migration of neural stem cells in hippocampus; reduces the number of microglial cell branches and terminal fingers, and inhibits neuroinflammation; increases the number of astrocytes and extends their terminal fingers, and strengthens the neuroprotection and support; increases the phosphorylation level of AKT in hippocampus, and activates the PI3K / AKT signaling pathway.

[0006] The 4-isopropyl toluene plays a significant role in anti-depression by inhibiting neuroinflammation, enhancing the support function of glial cells and activating the AKT signaling pathway, and has the potential to be developed as a new anti-depression candidate drug.

[0007] Therefore, the first object of the application is to provide the use of 4-isopropyl toluene or a pharmaceutically acceptable salt thereof in the preparation of an anti-depression drug.

[0008] Preferably, the 4-isopropyl toluene plays an anti-depression role through at least one of the following mechanisms: promoting the proliferation and migration of neural stem cells in hippocampus; reducing the number of microglial cell branches and terminal fingers, and inhibiting neuroinflammation; increasing the number of astrocytes and extending their terminal fingers, and strengthening the neuroprotection and support; increasing the phosphorylation level of AKT in hippocampus, and activating the PI3K / AKT signaling pathway.

[0009] Preferably, the drug contains an effective treatment amount of 4-isopropyl toluene or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable adjuvant.

[0010] Preferably, the pharmaceutically acceptable adjuvant is at least one selected from diluents, flavoring agents, binders, fillers, lubricants, wetting agents, disintegrants, solubilizers, solubilizers, pH regulators, osmotic pressure regulators, antioxidants, chelating agents, freeze-drying protectants.

[0011] Preferably, the administration route of the drug includes oral administration, sublingual administration, transdermal administration, injection administration or inhalation administration.

[0012] Preferably, the dosage form of the drug includes any one of tablets, capsules, granules, bulk powders, injections or freeze-dried powder injections.

[0013] It is a second object of the present application to provide an antidepressant medicament comprising a therapeutically effective amount of 4-isopropyltoluene or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0014] The present application has the following beneficial effects:

[0015] The present application first discovers and confirms that the natural monoterpene compound 4-isopropyltoluene has clear and multi-level antidepressant activity. Specifically, at the whole behavioral level, it can reverse the anhedonia, despair behavior and anxiety-like phenotype induced by chronic restraint stress; at the neuroendocrine level, it can effectively inhibit the overactivation of the HPA axis and reduce the peripheral corticosterone level; at the cellular and molecular level, it promotes hippocampal neural stem cell proliferation and migration, remodels astrocyte-microglia homeostasis, inhibits neuroinflammation and enhances neuroprotection by activating the PI3K / AKT signaling pathway. Therefore, 4-isopropyltoluene is first identified as a new, multi-target, potential antidepressant candidate with rapid onset and good safety, providing a new chemical entity and mechanism for the intervention of depression. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 : Effects of 4-isopropyltoluene on the behavioral patterns of mice. A: Corticosterone content determination, n=3; B: sucrose preference test, n=4; C: tail suspension test, n=4; D: novel environment inhibits food intake test, n=4; (data are expressed as mean ± standard error, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, compared with the soybean oil control group).

[0017] Figure 2 : Multidimensional regulation of neural plasticity by 4-isopropyltoluene in mice. A: Effect of 4-isopropyltoluene on neural stem cell proliferation in the hippocampal region of mice, n=6; B: Effect of 4-isopropyltoluene on neural stem cell migration in the hippocampal region of mice, n=6; C: Effect of 4-isopropyltoluene on γ-aminobutyric acid neurons in the posterior cingulate cortex of model mice, n=4; (data are expressed as mean ± standard error, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, compared with the soybean oil control group).

[0018] Figure 3: The effect of 4-isopropyltoluene on the reactivity of hippocampal glial cells in model mice. A: The effect of 4-isopropyltoluene on the morphology of hippocampal microglial cells in model mice, n = 6; B: The effect of 4-isopropyltoluene on the number and morphology of hippocampal astrocytes in model mice, n = 6; (Data are expressed as mean ± standard error, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, compared with the soybean oil control group).

[0019] Figure 4 : The effect of 4-isopropyltoluene on the expression of CREB and AKT proteins in the hippocampus of model mice, n = 3; (Data are expressed as mean ± standard error, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, compared with the soybean oil control group). DETAILED DESCRIPTION

[0020] The following examples are further illustrations of the present application and are not intended to limit the present application.

[0021] Example 1

[0022] 1. Materials

[0023] Experimental drugs and reagents: Resveratrol (Aladdin, CAS#: 501-36-0), 4-isopropyltoluene (Aladdin, CAS#: 99-87-6), soybean oil (Aladdin, CAS#: 8001-22-7).

[0024] Mouse Cortisol / Adrenocorticotropin (CORT) ELISA Kit (Fancuo, F2061-A), 4% paraformaldehyde general tissue fixative (biosharp, BL539A), PBS buffer (Solebao, P1020), PBST buffer (Xi'an Aouruijing, AP0831), sucrose (Fumeng chemical), boric acid (Fumeng chemical), sodium chloride (Fumeng chemical), Mouse Anti-Brdu (Millipore, MAB3424), Mouse Anti-GAD67 (ABclonal, AP1208), Goat Anti-Iba1 (NOVUSBIO, NB100-1028), Chicken Anti-GFAP (Origene, AP31806P), CREB (Cell Signaling, 9197), Phospho-CREB (Cell Signaling, 9198), Pan-Akt (ABclonal, A18120), Phospho-Akt (ABclonal, AP0637), GAPDH (Proteintech, 60004-1), 10% AP (weigh 1 g of AP, add 10 mL of deionized water, and fully dissolve, after aliquot, store at -20 °C), 10 × TBS (weigh 80 g of NaCl, 30 g of Tris, add 800 mL of deionized water, and fully dissolve, adjust pH to 7.4, then use deionized water to constant volume to 1000 mL), 1 × TBS (take 1000 mL of 10 × TBS, add 900 mL of deionized water, and add 1 mL of Tween-20, stir with a stirrer until completely mixed), 1 × electrophoresis liquid (weigh 14.413 g of Glycine, 3.028 g of Tris-base, 1.0 g of SDS, add 1000 mL of deionized water), 1 × transfer liquid (weigh 14.413 g of Glycine, 3.028 g of Tris-base, add 100 mL of methanol, and add 800 mL of deionized water, stir with a stirrer until completely mixed).

[0025] Experimental instruments: noise detector (Dell, DL33320i), freezing microtome (Leica, CM1900), 37°C incubator (Shanghai Yiheng, DHP-9082), centrifuge (Zhongjia, HC-3018R), different specifications of pipette (Dalong), water bath (Shanghai Qixin, DHG-9240A), laser confocal microscope (Zeiss, LSM800), ice maker (Xuexue, NP-IMS-60), refrigerator (Hisense, BCD-829WTVBP), horizontal shaker (Qislinbail, TS-200), electronic balance (Shimadzu, TW223L), electrophoresis instrument (Beijing Liu Yi Biological Technology Co., Ltd., DYY-6C), developing instrument (Tanon, Tanon 2500), enzyme-linked immunoassay instrument (Hangzhou Aosheng Instrument Co., Ltd., FlexA-200).

[0026] Experimental animals: 32 male Kunming (KM) mice [40±5g, 8 weeks old] were purchased from Shaanxi Xinchenglongyuan Biological Technology Co., Ltd.

[0027] 2. Method

[0028] Animal grouping, modeling and drug administration: 8-week-old male Kunming mice were fed under the condition of free intake of water and food, with 24h normal circadian rhythm. The mice were divided into normal control group (n=8) and depression modeling group (n=24). Chronic restraint stress method (Chronic Restraint Stress, CRS) was used to construct the depression model. After the success of the depression model, the depressed mice were randomly divided into 3 groups, 8 mice in each group, which were resveratrol treatment experimental group, 4-isopropyl toluene treatment experimental group and control group respectively. The experimental groups were treated with 50mg / kg of resveratrol, 4-isopropyl toluene (dissolved in soybean oil respectively). The control group was given the same dose of soybean oil, and the drug was given by intraperitoneal injection.

[0029] Sucrose preference test: Sucrose preference test assesses the reward response of rodents by double bottle selection method, measures the intake ratio of sucrose solution and pure water (SPI=sucrose intake / total liquid x 100%), which is used for depression model construction and antidepressant efficacy evaluation.

[0030] Tail suspension test: The tail of the mouse is fixed to make it hang upside down, and the still time after struggling is recorded to reflect the behavior of despair.

[0031] Novelty suppressed feeding test: By observing the phenomenon of reduced food intake of mice in a novel environment, the neural correlation between stress response and food intake regulation is revealed, and the food intake latency is used as an index to evaluate the antidepressant effect of drugs.

[0032] Corticosterone hormone content determination: The mouse corticosterone / adrenal hormone (CORT) ELISA research kit was used. The corticosterone specific antibody binds to the corticosterone in the sample, and the enzyme-labeled secondary antibody is used for color development. The absorbance (OD value) is measured to quantify the concentration of corticosterone.

[0033] Immunofluorescence staining: After the brain tissue was fixed with 4% paraformaldehyde solution and sucrose gradient sedimentation, 30 μm thick frozen sections were prepared. The primary antibody was prepared with 0.3% PBST and 2% fetal bovine serum: mouse DCX (1:500), mouse GAD67 (1:500), mouse BrdU (1:500), goat Iba1 (1:1000), chicken GFAP (1:1000) and 4°C overnight.

[0034] Western blot: The brain tissue of each group of mice was extracted, and the hippocampal protein was collected. The protein concentration was determined by BCA protein detection kit. 25 μg of total protein was loaded into a 10% gel and subjected to gel electrophoresis. The gel was transferred to a PVDF membrane, and the primary antibody rabbit CREB (1:1000), rabbit Phospho-CREB (1:1000), rabbit Pan-AKT (1:1000), rabbit Phospho-AKT (1:1000), mouse GAPDH (1:10000) was incubated at 4°C overnight. After TBST washing, the secondary antibody IgG (1:10000) was added and incubated at room temperature. The pictures were collected by Tanon 2500 system. Image J was used to statistically analyze the gray value of the target band, and the ratio of the target band to its corresponding internal standard was standardized.

[0035] Statistical analysis: The data of this study were analyzed and processed by GraphPad Prism 9.0 software. t-test and One way ANOVA were used for intergroup difference analysis, and the statistical results were expressed as mean ± SEM. P<0.05 was considered to have significant statistical difference (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

[0036] 3. Results

[0037] (1) Regulation of 4-isopropyltoluene on depressive-like behavior

[0038] The tail suspension test is commonly used to assess depressive-like behavior in mice and rats. In this study, the tail suspension test was used to evaluate whether the depression model was successfully constructed. The results showed that the resting time of the model group mice was significantly higher than that of the normal control group, indicating that the depression level of the model group mice was significantly higher than that of the normal control group, i.e., the modeling was successful. The subsequent experiment selected the successfully modeled mice as the depression-like model animals for this experiment. Corticosteroids are a class of steroid hormones synthesized and secreted by the adrenal cortex. There is a certain correlation between corticosteroids and depression. Studies have shown that the secretion level of corticosteroids may increase under long-term chronic stress or pressure, which is related to the occurrence and development of depression. Excessive corticosteroids can have a negative impact on the central nervous system, leading to changes in mood and cognitive function, thereby increasing the risk of depression. High levels of corticosteroids can affect neurotransmitter systems, such as reducing serum serotonin levels, which is one of the neurobiological mechanisms of depression. We evaluated the effect of 4-isopropyltoluene on depressive behavior in mice by detecting the content of corticosteroids in the blood of mice treated with 4-isopropyltoluene. The results showed Figure 1 A), the content of corticosteroids in the blood of mice in the 4-isopropyltoluene and resveratrol groups was significantly lower than that in the soybean oil control group. This indicates that 4-isopropyltoluene and resveratrol can reduce the content of corticosteroids and alleviate the depression of mice. As shown in Figure 1 B, the sucrose preference test results showed that the sucrose preference of mice in the 4-isopropyltoluene and resveratrol groups was significantly higher than that in the soybean oil control group, especially after 4-isopropyltoluene treatment, which effectively improved the sucrose preference of model mice, and the effect was better than that of resveratrol. The results of the tail suspension test Figure 1 C showed that the resting time of mice in the resveratrol group was significantly reduced, and the resting time of mice in the 4-isopropyltoluene group was decreased compared to the soybean oil control group, indicating that resveratrol and 4-isopropyltoluene can effectively alleviate the depressive behavior of model mice. Figure 1 D, the feeding latency of mice in the 4-isopropyltoluene and resveratrol groups was lower than that in the soybean oil control group, indicating that 4-isopropyltoluene and resveratrol can effectively alleviate the depressive behavior of model mice. The results of the behavioral paradigm showed that 4-isopropyltoluene had an effect on alleviating depressive-like behavior in model mice.

[0039] (2) Regulation of hippocampal neurogenesis by 4-isopropyltoluene in depressive mice

[0040] The hippocampus is a key brain region involved in memory, learning, and emotional regulation, and plays an important role in the pathogenesis of depression. Hippocampal volume reduction is associated with stress and depression, and hippocampal neurogenesis can buffer stress response and depressive behavior. BrdU was used to stain mouse hippocampal brain tissue sections. As shown in Figure 2As shown in A, the total number of positive cells in the 4-isopropyltoluene and resveratrol groups was significantly higher than that in the soybean oil control group, and the 4-isopropyltoluene group had a very significant difference, indicating that 4-isopropyltoluene had an improving effect on the depressive behavior of mice. Doublecortin (DCX) is a specific marker for immature neurons, and its expression level is often used to assess the activity of hippocampal dentate gyrus neurogenesis. Studies have shown that antidepressants can improve depressive behavior by promoting neurogenesis. In this study, the hippocampus of mice treated with 4-isopropyltoluene and resveratrol was stained, and the results showed that Figure 2 B), the total number of positive cells in the 4-isopropyltoluene and resveratrol groups was significantly higher than that in the soybean oil control group, and the 4-isopropyltoluene significantly increased the number of DCX positive cells in the DG region (hippocampal dentate gyrus), indicating that 4-isopropyltoluene can increase the number of DCX in the brain of depressed mice and promote the migration of neural stem cells. GABA is the main inhibitory neurotransmitter in the central nervous system, involved in emotion regulation, cognition and stress response. At the same time, a large number of studies have shown that in patients with depression, the number and activity of GABAergic neurons in the prefrontal cortex, hippocampus and cingulate cortex are significantly decreased, and the patients further develop emotional regulation disorders and cognitive disorders. Through GAD67 staining to label GABAergic neurons in the posterior cingulate cortex, Figure 2 C) The results showed that compared with the soybean oil control group, the number of GABAergic neurons in the resveratrol group was significantly increased. This indicates that resveratrol promotes the increase of GABAergic neurons in the posterior cingulate cortex, balances neurotransmitters, and relieves excessive excitation of the nervous system.

[0041] The above experimental results show that 4-isopropyltoluene and resveratrol have different degrees of promotion effect on model mouse hippocampal neurogenesis, such as newborn neural stem cells, stem cell migration, and intermediate neurons, indicating that 4-isopropyltoluene can improve the depressive behavior of model mice by remodeling neurogenesis.

[0042] (3) Changes in the morphology of glial cells in model mice

[0043] In the pathological process of depression, neuroinflammation often occurs, which can lead to changes in the number and morphology of glial cells, decrease in brain serotonin content, activation of hypothalamic-pituitary-adrenal axis, and impairment of synaptic plasticity and neurotransmission. Studies have shown that in rodent models of depression, the number and density of hippocampal astrocytes are more significantly reduced than neurons, and the total length of astrocyte processes in the hippocampus and prefrontal cortex of mice induced by chronic stress is shortened. In mice induced by chronic restraint stress (CRS), microglia in the prefrontal cortex and hippocampus of mice are in different degrees of over-activation state, and the number of microglia in the DG region of the hippocampus of mice is negatively correlated with the number of newborn neurons under chronic stress. In this experiment, the hippocampal DG region of mice treated with 4-isopropyl toluene and resveratrol was stained to find that (as shown in Figure 3 ), 4-isopropyl toluene and resveratrol can both reduce the number of microglia terminal end points to different degrees, indicating that 4-isopropyl toluene and resveratrol can improve depressive symptoms by reducing microglia activity, and 4-isopropyl toluene is better than resveratrol in effect Figure 3 A). At the same time, the results of astrocyte immunofluorescence staining show that the number of hippocampal dentate gyrus astrocytes in the 4-isopropyl toluene group is significantly increased compared with the control group, and the number of terminal end points is significantly increased, increasing the branch complexity Figure 3 B). 4-isopropyl toluene activates astrocytes to promote neural network integration and release anti-inflammatory factors to improve depressive symptoms.

[0044] (4) Expression of hippocampal depression-related proteins in model mice

[0045] Hippocampal neurogenesis can buffer stress response and depressive behavior. However, different stressors can lead to heterogeneity or even opposite stress responses in the hippocampus, such as the expression of plasticity-related proteins. Studies have shown that the expression of CREB in the hippocampus of patients with depression is down-regulated, and increasing the expression level of CREB can produce significant antidepressant-like behavior. In this study, the expression levels of CREB and Phospho-CREB in the hippocampus of mice treated with 4-isopropyl toluene and resveratrol were detected. The results showed that Figure 4), compared with the soybean oil control group, the expression level of Phospho-CREB in the 4-isopropyl toluene experimental group was significantly increased, and when CREB was activated, BDNF (brain-derived neurotrophic factor) expression was up-regulated, BDNF activated the MAPK pathway by binding to the TrkB receptor, and promoted the proliferation of NSCs. The AKT signaling pathway is the intersection of multiple signaling pathways, and the AKT signaling pathway plays a key role in the survival, growth and repair of neurons. The increase in the level of phosphorylation indicates that the activity of AKT is enhanced, which may help to promote the survival and repair of neurons and reduce neuronal apoptosis. For example, studies have found that by activating the PI3K-Akt signaling pathway, hippocampal neuronal apoptosis can be effectively reduced and its regeneration can be promoted, increasing hippocampal synaptic plasticity, thereby relieving depression. In this study, the expression levels of AKT and Phospho-AKT in the hippocampus of depressed mice treated with 4-isopropyl toluene and resveratrol were detected. The results showed that compared with the soybean oil control group, the expression level of AKT phosphorylation in the 4-isopropyl toluene experimental group was significantly increased, and AKT phosphorylation activated downstream target points such as GSK-3β, regulated the stability of cytoskeletal protein β-catenin, and promoted the directional migration of neural stem cells. The above results all show that 4-isopropyl toluene affects downstream signaling pathways by activating different protein phosphorylation, thereby promoting the proliferation and migration of neural stem cells, and thus improving the reduction of neurogenesis and impaired synaptic plasticity in depression.

[0046] The above detailed description is a specific description of the embodiments of the present application, which is not used to limit the patent scope of the present application, and any equivalent implementation or change without departing from the present application shall be included in the patent scope of the present application.

Claims

1. Use of 4-isopropyltoluene or a pharmaceutically acceptable salt thereof in the preparation of an antidepressant drug.

2. Use according to claim 1, characterized in that, The 4-isopropyltoluene exerts antidepressant effect by at least one of the following mechanisms: promoting hippocampal neural stem cell proliferation and migration; reducing the number of microglial cell branches and terminal processes, and inhibiting neuroinflammation; increasing the number of astrocytes and extending their terminal processes, and strengthening neuroprotection and support; and increasing the phosphorylation level of AKT in the hippocampus, and activating the PI3K / AKT signaling pathway.

3. Use according to claim 1, characterized in that, The drug comprises a therapeutically effective amount of 4-isopropyltoluene or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

4. Use according to claim 3, characterized in that, The pharmaceutically acceptable excipient is selected from at least one of diluents, flavoring agents, binders, fillers, lubricants, wetting agents, disintegrants, cosolvents, solubilizers, pH adjusters, osmotic pressure adjusters, antioxidants, chelating agents, and lyophilization protectants.

5. The use according to claim 1, characterized in that, The administration route of the drug includes oral administration, sublingual administration, transdermal administration, injection administration, or inhalation administration.

6. Use according to claim 1, characterized in that, The dosage form of the drug includes any one of tablets, capsules, granules, bulk powders, injections, or lyophilized powder injections.

7. An antidepressant medicament, characterized by comprising a compound of the formula (I) or a pharmaceutically acceptable salt thereof. The drug comprises a therapeutically effective amount of 4-isopropyltoluene or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

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