Application of brain-strengthening pill in preparation of medicine for treating depression

Abstract

The invention discloses application of a brain-strengthening pill in preparation of a medicine for treating depression. The brain-strengthening pill is prepared from 20-35 parts of radix angelicae sinensis, 10-15 parts of tabasheer, 20-27 parts of herba cistanche, 10-15 parts of dens draconis, 20-27 parts of rhizoma dioscoreae, 10-15 parts of amber, 15-21 parts of fructus schizandrae, 5-8 parts of rhizoma gastrodiae, 4-6 parts of semen boitae, 5-8 parts of radix salviae miltiorrhizae, 15-21 parts of fructus alpiniae oxyphyllae, 5-8 parts of radix ginseng, 10-15 parts of radix polygalae, 5-8 parts of flos chrysanthemi and 10-15 parts of rhizoma anemones altaicae. , 7-11 parts of ochre, 10-15 parts of arisaema cum bile, 40-55 parts of semen ziziphi spinosae, and 20-27 parts of fructus lycii. The brain strengthening pill can improve depression symptoms from multiple mechanisms, shows a good prospect of clinical application, is further deeply studied, such as application of research methods of multiple omics and the like, defines effective components and action targets and action mechanisms of the effective components, and is of great significance to clinical popularization and new drug development.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and particularly relates to the application of brain-enhancing pills in preparing medicines for treating depression. Background Art

[0002] Depression is a major disease that damages human health. It causes the most years of healthy life lost among all diseases and also creates a serious economic burden. Therefore, the treatment of depression has very important social significance and economic value.

[0003] There are many hypotheses for the pathogenesis of depression. The monoamine neurotransmitter hypothesis believes that the lack of monoamine neurotransmitters (5-HT, NE, DA) is the main mechanism of depression. Depressive symptoms can be improved by increasing the concentration of intersynaptic monoamine transmitters. Abnormal activation of the HPA axis is also one of the important mechanisms of depression. Stress can lead to increased secretion of CRH in the hypothalamus, and the secretion of ACTH in the pituitary gland will also increase in response to increased CRH secretion, and further lead to increased secretion of glucocorticoids in the adrenal cortex. Abnormal increase in glucocorticoids can further lead to mood changes, nerve cell damage and changes in neurotransmitter function, and cause depression. The neuroinflammation hypothesis is also one of the main mechanisms of depression. More and more studies have shown that serum IL-1β, IL-6, TNF-α and other proinflammatory cytokines are increased in patients with depression. NLRP3 inflammasomes play a very important role in neuroinflammation associated with depression. Stress can lead to reduced BDNF production. The serum BDNF level of patients with depression is reduced, but it can be restored after antidepressant treatment, indicating that BDNF is an important biomarker for depression, providing clinical support for the neurotrophic factor hypothesis of depression. Stress-induced ROS generation and excessive glucocorticoid production may be important causes of depression-related neuronal apoptosis.

[0004] Traditional Chinese medicine has good clinical efficacy and satisfactory safety in the treatment of depression, and can exert antidepressant effects through multiple mechanisms. It has great potential in the development of antidepressant drugs.

[0005] Jiannao Wan (JNW) is composed of 19 herbs, including Angelica sinensis, Radix dasyphyllae, Cistanche deserticola (salt-fried), Dragon's Tooth (calcined), Chinese yam, amber, Schisandra chinensis (steamed with wine), Gastrodia elata, Platycladus orientalis (fried), Salvia miltiorrhiza, Alpinia oxyphyllae (fried with salt), Ginseng, Polygala tenuifolia (fried with liquorice), Chrysanthemum, Acorus calamus, Ochre, Arisaema cinerea, Ziziphus jujubae seeds (fried with salt), and Lycium barbarum. It has the effects of strengthening the brain, improving intelligence, nourishing the heart and calming the nerves. It is used for overuse of the brain, memory loss, neurasthenia, dizziness, insomnia, dreaminess, restlessness, fatigue, fear of cold and weakness, and back pain. However, the antidepressant effect and mechanism of Jiannao Wan have not been systematically studied and verified. Summary of the invention

[0006] In view of the deficiencies of the prior art, the present invention provides the use of Jiannao Pills in the preparation of drugs for treating depression. The research of the present invention shows that Jiannao Pills can improve the symptoms of depression from multiple mechanisms, showing good prospects for clinical application.

[0007] The brain-strengthening pill contains 20-35 parts of angelica sinensis, 10-15 parts of rhizoma dahliae, 20-27 parts of cistanche deserticola, 10-15 parts of dragon's tooth, 20-27 parts of yam, 10-15 parts of amber, 15-21 parts of schisandra chinensis, 5-8 parts of gastrodia elata, 4-6 parts of cypress seed, 5-8 parts of salvia miltiorrhiza, 15-21 parts of alpinia oxyphylla, 5-8 parts of ginseng, 10-15 parts of polygala tenuifolia, 5-8 parts of chrysanthemum, 10-15 parts of calamus, 7-11 parts of ochre, 10-15 parts of rhizoma arisaematis, 40-55 parts of spiny jujube seed and 20-27 parts of wolfberry.

[0008] In the embodiments of the present invention, Jiannao Pills are provided as a drug for treating depression, and the dosage is 0.03 g / kg to 0.15 g / kg.

[0009] Furthermore, the dosage of Jiannao Pills as a drug for treating depression is 0.0375 g / kg to 0.15 g / kg.

[0010] In the embodiments of the present invention, there is provided the use of Jiannao Pills in the preparation of a drug for increasing the concentration of 5-HT and NE in hippocampal tissue and serum.

[0011] In the embodiments of the present invention, there is provided the use of Jiannao Pills in the preparation of a drug for reducing the levels of hypothalamic CRH and serum ACTH and CORT.

[0012] In an embodiment of the present invention, there is provided the use of Jiannao Pills in the preparation of a drug for reducing the contents of IL-1β, IL-6 and TNF-α in serum and hippocampal tissue.

[0013] In an embodiment of the present invention, there is provided the use of Jiannao Pills in preparing a drug for reducing the expression of NLPR3 mRNA in hippocampal tissue.

[0014] In an embodiment of the present invention, there is provided the use of Jiannao Pills in preparing a drug for restoring the normal expression of NLPR3 and Caspase-1 proteins in hippocampal tissue.

[0015] In the embodiments of the present invention, there is provided the use of Jiannao Pills in preparing a drug for up-regulating the expression of BDNF and TrκB.

[0016] In the embodiments of the present invention, there is provided the use of Jiannao Pills in preparing a drug for up-regulating the expression of Bcl-2 mRNA and reducing the expression of Bax mRNA.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) The present invention provides the use of Jiannao Pills in the preparation of drugs for treating depression. The effect of Jiannao Pills on improving depressive symptoms was studied using CUMS model mice, and its mechanism of action was explored, providing a theoretical basis for the clinical treatment of depression with Jiannao Pills.

[0019] (2) For CUMS model mice, Jiannao Pills can improve the fur condition of model mice in a dose-dependent manner, restore sucrose preference, increase the number, time and movement distance of entering the central area in the open field test, and reduce the immobility time in the tail suspension test and forced swimming test. There are significant differences compared with the model group, indicating that Jiannao Pills can improve the behavioral symptoms of depression.

[0020] (3) The present invention found that Jiannao Pills can simultaneously increase the levels of 5-HT and NE in the hippocampus and serum of model mice; can improve the pathological changes of "abnormal activation of the HPA axis in CUMS model mice, with significantly higher levels of hypothalamic CRH and serum ACTH and CORT than those in normal mice"; can dose-dependently reduce the levels of IL-1β, IL-6, and TNF-α in the serum and hippocampus of CUMS mice, and at the same time, can significantly downregulate the mRNA transcription levels of IL-1β, IL-6, and TNF-α in the hippocampus, suggesting that it can reduce neuroinflammation; can significantly reduce the upregulation of NLRP3 mRNA and protein expression in mice caused by CUMS, can reduce the IL-1β mRNA expression in the hippocampus of model mice and the IL-1β content in serum and hippocampus, and may interfere with the formation of NLRP3 inflammasomes and their Caspase-1 activation function, thereby regulating the production of IL-1β and exerting an antidepressant effect.

[0021] (3) The present study found that the IκBα content in the hippocampus of CUMS model mice was significantly reduced, while the p-IκBα content was significantly increased, and the p-IκBα / IκBα ratio was significantly increased, indicating that the NF-κB signaling pathway was activated. Jiannao Pills can reduce the p-IκBα content and increase the IκBα content in model mice, suggesting that Jiannao Pills can effectively inhibit the activation of the NF-κB signaling pathway.

[0022] (4) The present invention shows that Jiannao Pills can improve the symptoms of depression through multiple mechanisms, showing good prospects for clinical application. Further in-depth research on it, such as the use of multi-omics and other research methods to clarify the effective ingredients and their targets and mechanisms of action, is of great significance for its clinical promotion and new drug development.

[0023] (5) The present study shows that fluoxetine, as a selective 5-hydroxytryptamine (5-HT) reuptake inhibitor, can significantly increase the concentration of 5-HT in the hippocampus and serum of CUMS model mice, but has no significant effect on NE, while Jiannao Pills have a certain effect on increasing the concentration of NE in the hippocampus and serum. The Jiannao Pills in each dose group have a better effect on reducing the levels of IL-1β, IL-6, and TNF-α in the serum and hippocampus of mice than fluoxetine. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0025] Figure 1 The effect of Jiannao Pills on the body weight of CUMS model mice in the example. Note: Compared with the normal group, * indicates P < 0.05, ** indicates P < 0.01;

[0026] Figure 2 The effect of Jiannao Pills on the fur status of CUMS model mice in the example. Note: Compared with the normal group, △ indicates P < 0.05, △△ Indicates P < 0.01; compared with the model group, * indicates P < 0.05, ** indicates P < 0.01;

[0027] Figure 3 The effect of Jiannao Pills on sucrose preference of CUMS model mice in the example. Note: Compared with the normal group, △ indicates P < 0.05, △△ Indicates P < 0.01; compared with the model group, * indicates P < 0.05, ** indicates P < 0.01;

[0028] Figure 4 The effect of Jiannao Pills on the immobility time of tail suspension in CUMS model mice in the examples. Note: Compared with the model group, * indicates P < 0.05, ** indicates P < 0.01;

[0029] Figure 5 The effect of Jiannao Pills on the immobility time of forced swimming in CUMS model mice in the examples. Note: Compared with the model group, * indicates P < 0.05, ** indicates P < 0.01;

[0030] Figure 6 The effect of Jiannao Pills on the behavior of CUMS model mice in the open field test in the example, where (a) is the number of times the mouse enters the central area of ​​the open field, (b) is the time the mouse stays in the central area, and (c) is the distance moved; Note: Compared with the model group, * indicates P < 0.05, and ** indicates P < 0.01;

[0031] Figure 7 The effects of Jiannao Pills on neurotransmitters in CUMS model mice in the examples, where (a) is the 5-HT concentration in mouse hippocampus, (b) is the 5-HT concentration in mouse serum, (c) is the NE concentration in mouse hippocampus, and (d) is the NE concentration in mouse serum; Note: Compared with the model group, * indicates P < 0.05, and ** indicates P < 0.01;

[0032] Figure 8 The effect of Jiannao Pills on the HPA axis of CUMS model mice in the examples, where (a) is the CRH level in the hypothalamus of mice, (b) is the ACTH level in the serum of mice, and (c) is the CORT level in the serum of mice; Note: Compared with the model group, * indicates P < 0.05, ** indicates P < 0.01;

[0033] Fig. 9 The effects of Jiannao Pills on cytokine secretion in CUMS model mice in the examples, wherein (a) is the IL-1β content in mouse serum, (b) is the IL-6 content in mouse serum, (c) is the TNF-α content in mouse serum, (d) is the IL-1β content in mouse hippocampal tissue, (e) is the IL-6 content in mouse hippocampal tissue, and (f) is the TNF-α content in mouse hippocampal tissue; Note: compared with the model group, * indicates P<0.05, and ** indicates P<0.01;

[0034] Fig.10 The effect of Jiannao Pills on cytokine mRNA expression in CUMS model mice in the examples, where (a) is the IL-1β expression level in mouse hippocampus tissue, (b) is the IL-6 expression level in mouse hippocampus tissue, and (c) is the TNF-α mRNA expression level in mouse hippocampus tissue; Note: Compared with the model group, * indicates P < 0.05, ** indicates P < 0.01;

[0035] Fig.11 The effect of Jiannao Pills on NLRP3 mRNA expression in the hippocampus of CUMS model mice in the example. Note: Compared with the model group, * indicates P < 0.05, ** indicates P < 0.01;

[0036] Fig.12 The effect of Jiannao Pills on the expression of Caspase-1 mRNA in the hippocampus of CUMS model mice in the example. Note: Compared with the model group, * indicates P < 0.05, ** indicates P < 0.01;

[0037] Fig.13The effect of Jiannao Pills on the expression of proteins in the NF-κB / NLRP3 signaling pathway of CUMS model mice in the examples, wherein (a) is the expression graph of each protein, (b) is the relative expression level graph of NF-κB protein, (c) is the relative expression level graph of IκBα protein, (d) is the relative expression level graph of phosphorylated IκBα protein, (e) is the relative expression level graph of NLPR3 protein, and (f) is the relative expression level graph of Caspase-1 protein. Note: Compared with the model group, * indicates P < 0.05, and ** indicates P < 0.01;

[0038] Fig.14 The effect of Jiannao Pills on BDNF mRNA expression in CUMS model mice in the example. Note: Compared with the model group, * indicates P < 0.05, ** indicates P < 0.01;

[0039] Fig.15 The effect of Jiannao Pills on TrκB mRNA expression in CUMS model mice in the example. Note: Compared with the model group, * indicates P < 0.05, ** indicates P < 0.01;

[0040] Fig.16 The effect of Jiannao Pills on the expression of BDNF and TrκB proteins in CUMS model mice in the examples, where (a) is a protein expression graph, (b) is a graph of the relative expression level of BDNF protein, and (c) is a graph of the relative expression level of TrκB protein. Note: Compared with the model group, * indicates P < 0.05, and ** indicates P < 0.01;

[0041] Fig.17 The effect of Jiannao Pills on Bcl-2 mRNA expression in CUMS model mice in the examples. Note: Compared with the model group, * indicates P < 0.05, ** indicates P < 0.01;

[0042] Fig.18 The effect of Jiannao Pills on Bax mRNA expression in CUMS model mice in the example. Note: Compared with the model group, * indicates P < 0.05, ** indicates P < 0.01;

[0043] Fig.19 The effect of Jiannao Pills on the expression of Bcl-2 and Bax proteins in CUMS model mice, where (a) is the protein expression graph, (b) is the relative expression level graph of Bcl-2 protein, and (c) is the relative expression level graph of Bax protein. Note: Compared with the model group, * indicates P < 0.05, and ** indicates P < 0.01. DETAILED DESCRIPTION

[0044] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0045] The experimental methods in the following examples are conventional methods unless otherwise specified. The medicinal materials, reagents, etc. used in the following examples are commercially available products unless otherwise specified.

[0046] In the examples of the present invention, the intervention effect of Jiannao Pills on CUMS depression model mice was studied, and its mechanism of action was explored.

[0047] The ingredients of the brain-boosting pills are cited from the prior patent "A method for preparing brain-boosting pills" (publication number: CN110478456A), including 20-35 parts of angelica sinensis, 10-15 parts of rhizoma dahliae, 20-27 parts of cistanche deserticola, 10-15 parts of dragon teeth, 20-27 parts of yam, 10-15 parts of amber, 15-21 parts of schisandra chinensis, 5-8 parts of gastrodia elata, 4-6 parts of cypress seed, 5-8 parts of salvia miltiorrhiza, 15-21 parts of alpinia oxyphylla, 5-8 parts of ginseng, 10-15 parts of polygala tenuifolia, 5-8 parts of chrysanthemum, 10-15 parts of calamus, 7-11 parts of ochre, 10-15 parts of arisaema ciliata, 40-55 parts of spiny jujube seed, and 20-27 parts of wolfberry.

[0048] The formula of the brain-strengthening pill used in the embodiment of the present invention: 25 parts of angelica, 10 parts of dahlia, 20 parts of cistanche, 10 parts of dragon teeth, 20 parts of yam, 10 parts of amber, 15 parts of schisandra, 5 parts of gastrodia, 4 parts of pine nuts, 5 parts of salvia miltiorrhiza, 15 parts of alpinia oxyphylla, 5 parts of ginseng, 10 parts of polygala, 5 parts of chrysanthemum, 10 parts of calamus, 7.5 parts of ochre, 10 parts of arisaema, 40 parts of jujube seeds, and 20 parts of wolfberry.

[0049] Chronic Unpredictable Mild Stress (CUMS) is a depression research method widely used in animal experiments. By applying a series of unpredictable mild stressors to experimental animals, this model can induce behavioral and physiological changes similar to depression, thereby helping researchers better understand the pathogenesis of depression and evaluate the effectiveness of drug treatment.

[0050] Experimental study on the effects of Jiannao Pills on CUMS depression model mice

[0051] 1. Research Materials

[0052] 1.1 Experimental animals

[0053] Balb / c mice, 48 all males, 8 weeks old, weighing 18-22 g, were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd. Balb / c mice were raised in the SPF animal laboratory of the Animal Experiment Center of Sichuan Academy of Traditional Chinese Medicine.

[0054] 1.2 Trial Drugs

[0055] Brain-enhancing pills, 10 pills weighing 1.5g each, 400 pills / bottle, national medicine standard Z37020791, Shanghai Pharmaceutical Group Qingdao Guofeng Pharmaceutical Co., Ltd., product batch number: 1721012. Fluoxetine hydrochloride capsules, 20mg / pill, 28 pills / box, national medicine standard H20073985, Shanxi Qianyuan Pharmaceutical Group Co., Ltd., product batch number: 230602.

[0056] 1.3 Test reagents

[0057] Pentobarbital sodium, Sinopharm Chemical Reagent Co., Ltd., batch number: WS20051129.

[0058] Mouse 5-hydroxytryptamine (5-HT) ELISA kit (Cat. No.: MM-0443M1), mouse norepinephrine (NE) ELISA kit (Cat. No.: MM-0876M1), mouse adrenocorticotropic hormone (ACTH) ELISA kit (Cat. No.: MM-0554M2), mouse corticotropin-releasing hormone (CRH) ELISA kit (Cat. No.: MM-0509M2), mouse corticosterone (CORT) ELISA kit (Cat. No.: MM-0061M2), mouse norepinephrine (NE) ELISA kit (catalog number: MM-0627M1), mouse interleukin 1β (IL-1β) ELISA kit (catalog number: MM-0040M1), mouse interleukin 6 (IL-6) ELISA kit (catalog number: MM-0163M1), mouse tumor necrosis factor α (TNF-α) ELISA kit (catalog number: MM-0132M1) were purchased from Jiangsu Enzyme Immunity Industry Co., Ltd. RNA extraction kit TransZol Up Plus RNA Kit (catalog number: ER501-01), first-strand cDNA synthesis kit Uni All-in-One First-Strand cDNASynthesis SuperMix for qPCR (One-Step gDNARemoval) (Cat. No.: AU341) was purchased from Beijing Quanshijin Biotechnology Co., Ltd. Real-time fluorescence quantitative PCR kit MagicSYBR Mixture (Cat. No.: CW3008) was purchased from Kangwei Century Biotechnology Co., Ltd.

[0059] Primers were purchased from Shanghai Sangon Biotechnology Co., Ltd. and synthesized. The primer sequences are shown in 2.4.3.4 qPCR detection.

[0060] RIPA lysis buffer (P0013B), phosphorylation protease inhibitor (P1081), BCA protein quantitative detection kit (P0012), 5× protein loading buffer (P0015) were purchased from Shanghai Bio-Tech Biotechnology Co., Ltd. PMSF (100 mM) (P0100) and SDS-PAGE gel preparation kit (P1200) were purchased from Beijing Solebao Technology Co., Ltd. Protein Marker (P1106) was purchased from Beijing Prilai Gene Technology Co., Ltd. NLRP3 (30109-1-AP), Caspase-1 (81482-1-RR), NF-κB p65 (80979-1-RR), IκBα (10268-1-AP), BDNF (28205-1-AP), TrkB (13129-1-AP), Bax (50599-2-Ig), Bcl-2 (26593-1-AP), β-actin (66009-1-Ig), HRP-Goat anti Rabbit (SA00001-2) were purchased from Proteintech. p-IκBα (AF2002) was purchased from Affinity.

[0061] 1.4 Test equipment

[0062] Small animal behavior recorder, model: EthoVision XT 8.5, manufacturer: Noldus Information Technology BV., the Netherlands. Automatic microplate reader, model: BIOBASE-EL10A, manufacturer: Shandong Bojin Medical Instrument Co., Ltd.

[0063] 2. Research Methods

[0064] 2.1 Animal grouping and drug administration

[0065] The mice were ear-tagged and adaptively fed for 1 week. During the adaptive feeding period, sucrose preference test training and baseline measurement were performed: the mice were housed individually in a single cage, and were given two bottles of 3% sucrose solution on the first day for adaptation, and one bottle of 2% sucrose solution and one bottle of distilled water on the second day for adaptation. On the third day, one bottle of 2% sucrose solution and one bottle of distilled water weighed in advance were given again, and the positions of the two water bottles were exchanged after 12 hours. The remaining weight of the two water bottles was recorded after 24 hours, and the consumption of the two liquids was calculated. Sucrose preference degree = sucrose solution consumption / (sucrose solution consumption + distilled water consumption)*100%.

[0066] According to the degree of sucrose preference and body weight, mice were randomly divided into 6 groups, 8 mice in each group, namely: normal group (Control), model group (Model), JNW high-dose group (JNW-H), JNW medium-dose group (JNW-M), JNW low-dose group (JNW-L), fluoxetine group (FLU). During the experiment, the normal group was kept 4 mice / cage, with unlimited feed and drinking water, and did not receive CUMS. The mice in the other groups were kept in single cages and received CUMS for 6 consecutive weeks.

[0067] After 2 weeks of CUMS, the animals in each group began to be medicated. The normal group and the model group were gavaged with distilled water, 0.1 mL / 10 g body weight daily; the JNW high-dose group, JNW medium-dose group, and JNW low-dose group were gavaged with JNW suspension at a concentration of 0.15 g / mL, 0.075 g / mL, and 0.0375 g / mL, respectively, at a concentration of 0.1 mL / 10 g body weight (the doses were 1.5 g / kg, 0.75 g / kg, and 0.375 g / kg, respectively, which were equivalent to 40 times, 20 times, and 10 times the clinical doses used in humans, respectively); the fluoxetine group was gavaged with fluoxetine suspension at a concentration of 1 mg / mL, 0.1 mL / 10 g body weight daily (the dose was 10 mg / kg, equivalent to 33 times the clinical dose used in humans), for 32 consecutive days.

[0068] 2.2 CUMS model preparation

[0069] Except for the mice in the normal group, the mice in the other groups received CUMS stimulation in a semi-random manner every week for 6 weeks. The mild stimulation was shown in Table 1. The weekly CUMS stimulation regimen is shown in Table 2.

[0070] Table 1 Mild stimulation types and descriptions

[0071] Serial number type illustrate 1 Food deprivation Fasting for 12 hours 2 water deprivation No water for 12 hours 3 Wet cage Add water to the empty breeding box to a depth of about 1 cm for 4 hours. 4 Tilted cage The breeding box is tilted 45° against the wall and kept overnight. 5 Dampened sawdust Add an appropriate amount of water to the litter to soak it all, and leave it overnight. 6 Rat bedding Add 60 mL of rat dirty bedding to the feeding box for 2 h. 7 Pair housing Randomly pair and place a stranger mouse in the room for 2 hours. 8 Cage change Randomly place the mouse in a box with another mouse overnight. 9 Physical restraint The mouse was placed in a 50 mL centrifuge tube (maintaining ventilation) for 2 h. 10 Cold Swim in 10℃ water bath for 5 min

[0072] Table 2 CUMS stimulation scheme

[0073] Monday Tuesday Wednesday Thursday Friday Saturday Sunday Physical limitations Pair breeding Wet breeding box Cold stimulation Pair breeding No water fasting Cold stimulation wet litter Rat bedding - Physical limitations Tilt feeding box Exchange Box

[0074] 2.3 Behavioral evaluation

[0075] 2.3.1 Fur evaluation

[0076] Before modeling every Monday, the fur was evaluated. At 9 a.m. on the test day, the fur of each mouse was scored on the head, neck, back, abdomen, tail, forelimbs, and hindlimbs. The scoring criteria were: smooth, shiny, neat, and tangle-free fur, 0 points; slightly messy, fluffy, with slight tangles, 0.5 points; obviously untidy, fluffy, and stained, 1 point, and the scores of the 7 parts were added up to the total score.

[0077] 2.3.2 Sucrose preference test

[0078] Sucrose preference test was performed every Thursday morning after modeling. Mice were housed in single cages and given 1 bottle of 2% sucrose solution and 1 bottle of distilled water weighed in advance. The positions of the two water bottles were exchanged after 12 hours. The remaining weight of the two water bottles was recorded after 24 hours, and the consumption of the two liquids was calculated. Sucrose preference degree = sucrose solution consumption / (sucrose solution consumption + distilled water consumption)*100%.

[0080] 2.3.3 Start and end motionless time

[0081] On the 29th day after administration, the tail of the mouse was taped to an iron stand and hung upside down, with the head 25 cm away from the table. The time was counted for 6 minutes, and the time the mouse remained motionless within the next 5 minutes was recorded.

[0082] 2.3.4 Forced swimming immobility time

[0083] On the 30th day after administration, the mice were placed in a 2L beaker and allowed to swim in water at 24-26°C for 6 minutes. The time during which the mice remained motionless within the next 5 minutes was recorded.

[0084] 2.3.5 Open field experiment

[0085] On the 31st day after administration, each mouse was placed in an open field test arena, and the time spent in the central area and the edge area and the movement distance of the mouse within 5 minutes were observed and recorded.

[0086] 2.4 Mechanism of action studies

[0087] 2.4.1 Sample preparation

[0088] 30 minutes after the last administration, mice were anesthetized by intraperitoneal injection of 60 mg / kg sodium pentobarbital. After the disappearance of flipping, the eyeballs of mice were pulled out to collect blood. After the blood was left to stand for 1 hour, it was centrifuged at 3000 rpm for 10 minutes, and serum was collected and stored at -80°C for later use. After the blood was collected from the mice, the brain tissue was quickly separated, and the hypothalamus and bilateral hippocampus were separated. The hypothalamus, left hippocampus, and right hippocampus of each mouse were placed in centrifuge tubes and stored at -80°C for later use.

[0089] 2.4.2ELISA test

[0090] 2.4.2.1 Sample processing

[0091] Serum: After the frozen serum is taken out, it is placed at room temperature to slowly thaw and return to room temperature before testing.

[0092] Tissue samples: Add PBS with a pH of about 7.2-7.4 to a centrifuge tube at a ratio of 1:9, and homogenize the specimen thoroughly with a homogenizer. Centrifuge at 3000rpm for 20min, and take the supernatant for testing.

[0093] 2.4.2.2 5-HT determination

[0094] The mouse 5-hydroxytryptamine (5-HT) ELISA kit was used to measure the level of 5-hydroxytryptamine according to the instructions.

[0095] 2.4.2.3NE determination

[0096] Mouse norepinephrine (NE) ELISA kit was used for determination according to the operating method described in the instruction manual.

[0097] 2.4.2.4CORT assay

[0098] Mouse corticosterone (CORT) ELISA kit was used to measure the level of CORT according to the instructions.

[0099] 2.4.2.5 ATCH assay

[0100] Mouse adrenocorticotropic hormone (ACTH) ELISA kit was used to measure the level of ACTH according to the instructions.

[0101] 2.4.2.6 CRH determination

[0102] Mouse corticotropin-releasing hormone (CRH) ELISA kit was used to measure the level of CRH according to the instructions.

[0103] 2.4.2.7 IL-1β assay

[0104] Mouse interleukin 1β (IL-1β) ELISA kit was used to measure the level of interleukin 1β according to the instructions.

[0105] 2.4.2.8 IL-6 assay

[0106] Mouse interleukin 6 (IL-6) ELISA kit was used to measure the TNF-α level according to the instructions.

[0107] Mouse tumor necrosis factor α (TNF-α) ELISA kit was used to measure the level of TNF-α according to the operating method described in the instruction manual.

[0108] 2.4.3 RT-PCR detection

[0109] 2.4.3.1 RNA extraction

[0110] (1) Take frozen hippocampal tissue and immediately add 1 mL of TransZol Up and homogenize it using a homogenizer.

[0111] (2) For every 1 ml of TransZol Up used, add 0.2 ml of chloroform, shake vigorously for 30 seconds, and incubate at room temperature for 3 minutes.

[0112] (3) Centrifuge at 10,000g for 15 minutes at 2-8℃. The sample is now divided into three layers: a colorless aqueous phase (upper layer), a middle layer, and a pink organic layer (lower layer). The RNA is in the aqueous phase, and the volume of the aqueous phase is about 50%-60% of the TransZol Up reagent used (in order to avoid DNA contamination caused by aspiration into the middle layer, a portion of the aqueous phase can be appropriately left).

[0113] (4) Transfer the colorless aqueous phase to a new centrifuge tube, add an equal volume of anhydrous ethanol (precipitate may appear at this time), and gently invert to mix.

[0114] (5) Add the resulting solution and precipitate to a centrifuge column and centrifuge at 12,000 g for 30 seconds at room temperature. Discard the flow-through (if the volume is larger than the capacity of the centrifuge column, this can be done in several steps).

[0115] (6) Add 500 μL CB9, centrifuge at 12,000 g for 30 seconds at room temperature, and discard the flow-through.

[0116] (7) Repeat step 6 once.

[0117] (8) Add 500 μL WB9 (confirm whether anhydrous ethanol has been added before use), centrifuge at 12,000 g for 30 seconds at room temperature, and discard the flow-through.

[0118] (9) Repeat step 8 once.

[0119] (10) Centrifuge at 12,000 g for 2 min at room temperature to completely remove residual ethanol.

[0120] (11) Place the spin column in the RNase-free Tube (provided in the kit), add 50-200 μL RNase-free water in the center of the spin column, and let it stand at room temperature for 1 minute.

[0121] (12) Centrifuge at 12,000 g for 1 minute at room temperature to elute the RNA. Store the RNA at -80°C.

[0122] 2.4.3.2 RNA detection

[0123] (1) Electrophoresis detection

[0124] Add 2 μL 2× RNA Loading Buffer to 3 μL sample and take appropriate amount of sample for detection. Electrophoresis parameters: gel concentration 1.2%, voltage 180V, electrophoresis 15min. Take photos and detect in gel imaging analyzer.

[0125] (2) Concentration detection:

[0126] Take 1 μL RNA and use Novogene ultra-micro spectrophotometer US850 to perform A260 / A280 and A260 / A230 detection.

[0127] 2.4.3.3 First-strand cDNA synthesis

[0128] After the concentration is normalized, perform first-strand cDNA synthesis and gDNA removal. Prepare the reaction system according to the table below and mix thoroughly. Centrifuge briefly, 42℃ for 15min, and terminate the reaction at 85℃ for 5s. Add the reverse transcription product to 180μL ddH2O and mix thoroughly for use.

[0129]

[0130] 2.4.3.4qPCR detection

[0131] (1) Primer information

[0132]

[0133] (2) qPCR preliminary experiment

[0134] Two samples were randomly selected to perform single-well qPCR detection of all genes, with NTC control. The reaction system is as follows:

[0135]

[0136] The amplification procedure is as follows:

[0137]

[0138] (3) Electrophoresis detection of qPCR pre-experimental products:

[0139] Take 2 μL of sample and add 2 μL of 6×DNA Loading Buffer, and take appropriate amount of sample for detection. Electrophoresis parameters: gel concentration 1%, voltage 120V, electrophoresis time 20min. Take photos and detect in gel imaging analyzer.

[0140] (4) Sample qPCR formal experiment

[0141] The primers successfully verified in the qPCR preliminary experiment were tested according to the preliminary experimental conditions, with three technical replicates for each reverse transcription product, and the reaction system and procedures were carried out according to the preliminary experimental conditions.

[0142] 2.4.3.5 qPCR data quality control and analysis

[0143] By testing whether the amplification curve presents a good S-shape, whether the melting curve has a single peak and a normal peak shape, and the data repeatability error of the repeated test CT value ≤33 is ≤0.5, the data that meets the above requirements can be used for subsequent data analysis.

[0144] Adoption 2 -ΔΔCT Methods Calculate the differential expression fold of the target gene, that is: A = CT (target gene of the test sample) - CT (internal standard gene of the test sample), B = CT (target gene of the control sample) - CT (internal standard gene of the control sample), differential expression fold = 2 -(A-B) .

[0145] 2.4.4 Western Blot Detection

[0146] 2.4.4.1 Total protein extraction

[0147] Take the frozen hippocampal tissue and cut it into small pieces. Add lysate at a ratio of 150-250 μL per 20 mg of tissue. Use a homogenizer to homogenize until fully lysed. Centrifuge at 12000 rpm and 4°C for 10 min, and collect the supernatant, which is the total protein solution.

[0148] The loading amount of each well is 10 μg protein, which can be increased according to the experimental requirements. According to the protein quantification results, take the required total protein solution and add an appropriate amount of loading buffer, boil it in water bath for 10 minutes, centrifuge it, and take the supernatant for loading.

[0149] 2.4.4.2 SDS-PAGE electrophoresis

[0150] (1) Prepare the glue making apparatus and assemble the glass plate for glue making.

[0151] (2) Take a tube of 500 mg AP powder and add it to 5 mL pure water to dissolve it into a 10% AP solution for later use. AP solution is easily lost, but it is effective for one week when stored at 4°C away from light. It is effective for one year when stored at -20°C after aliquoting.

[0152] (3) According to the number of glues to be made, take an appropriate amount of lower glue solution A and lower glue solution B and mix them to make lower glue preparation solution. It can be placed at 4°C and used within one day. Before pouring the lower glue, add 10% AP solution at a ratio of 10% AP solution: lower glue preparation solution = 1:100. After mixing, pour the glue immediately, then slowly move along the glass plate for making glue and add pure water to seal the lower glue to eliminate bubbles. After standing at room temperature for 30 minutes, the lower glue is completely solidified.

[0153] (4) While waiting for the lower layer of gel to solidify, you can prepare the upper layer of gel. According to the number of gels to be prepared, take an appropriate amount of upper layer gel solution A and upper layer gel solution B, mix them in equal volumes to prepare the upper layer gel preparation solution (a colored liquid), which can be stored at 4°C and used within one day.

[0154] (5) After the lower layer of glue is completely solidified, discard the pure water. Before pouring the upper layer of glue, add 10% AP solution at a ratio of 10% AP solution: upper layer glue preparation solution = 1:100. After mixing, pour the glue immediately and quickly insert the appropriate glue comb. After the upper layer of glue is completely solidified at room temperature for 30 minutes, slowly pull out the glue comb.

[0155] (6) Take the prepared gel plate and assemble the electrophoresis device.

[0156] (7) Add enough electrophoresis solution and load the sample for electrophoresis. Add the sample to the electrophoresis well and perform electrophoresis. The voltage of the concentrated gel is 75V and that of the separation gel is 100V. The electrophoresis can be terminated when the bromophenol blue just runs out and the membrane can be transferred.

[0157] 2.4.4.3 Transfer

[0158] (1) Prepare 6 pieces of 7×9 cm filter paper and a PVDF membrane of appropriate size. The PVDF membrane must be activated with methanol before use.

[0159] (2) Place transfer clips, two sponge pads, a glass rod, filter paper and activated PVDF membrane in a basin containing transfer solution.

[0160] (3) Open the clamps and keep the black side horizontal. Place a sponge and three layers of filter paper on the mat.

[0161] (4) Carefully peel off the separation gel and cover it on the filter paper. Cover the membrane on the gel and remove bubbles. Cover the membrane with three pieces of filter paper and remove bubbles. Finally, cover it with another sponge pad.

[0162] (5) Place the assembled sandwich into a transfer mold and transfer at 200 mA for 1 hour.

[0163] 2.4.4.4 Immune response

[0164] (1) Block the transferred membrane with 5% skim milk (prepared in TBST) on a decolorizing shaker at room temperature for 1 h.

[0165] (2) Add an appropriate volume of primary antibody diluent (prepared with 5% BSA solution dissolved in TBST; the dilution ratio is based on the recommendations in the primary antibody manual) and incubate at 4°C overnight.

[0166] (3) Wash the membrane three times with TBST on a decolorizing shaker at room temperature for 5 min each time.

[0167] (4) Add an appropriate volume of primary antibody diluent (diluted 5000 times with TBST 5% BSA solution) and incubate at room temperature for 30 min.

[0168] (5) Wash three times with TBST on a decolorizing shaker at room temperature, 5 min each time.

[0169] 2.4.4.5 Chemiluminescence

[0170] Mix the two reagents ECL-A and ECL-B in equal volumes in a centrifuge tube, place the PVDF membrane with the protein side facing up so that it is fully in contact with the mixture, then place the PVDF membrane in a tray, put it into an exposure instrument, select a suitable light source and aperture, preview the exposure for 30 seconds, and adjust the exposure time by 3 minutes to 10 minutes based on the preview image to obtain the exposure result.

[0171] 2.4.4.6 Gel Image Analysis

[0172] The images were numbered and archived, and after being sorted and adjusted using PhotoShop, the grayscale value of the protein bands was analyzed using ImageJ software.

[0173] 3. Research Results

[0174] 3.1 Effect of JNW on the body weight of CUMS model mice

[0175] The weight changes of mice during the experiment were as follows Figure 1 As shown. Before modeling, the weight of mice in each group was basically the same. As the modeling progressed, the weight gain of mice in the model group was significantly less than that in the normal group, and there was a significant difference between the two groups (P<0.05). Although the weight gain of each drug-treated group was higher than that of the model group, no significant difference was observed.

[0176] 3.2 Effect of JNW on the fur status of CUMS model mice

[0177] The fur state is a function of the frequency and degree of grooming behavior, so it can reflect the animal's self-centered activity motivation. Throughout the experiment, the fur of the mice in the normal group was smooth and tangle-free, and the fur was rarely messy or tangled; as the modeling time prolonged, the fur of the mice in the model group on the head, neck, abdomen and other parts gradually became messy, fluffy and stained. The fur score was significantly different from that of the normal group starting from the second week after modeling (P<0.01). Figure 2 As shown in the data, after administration of different doses of JNW and fluoxetine, the fur condition of the model mice was significantly improved. The fur scores of the JNW high-dose group and the fluoxetine group were significantly lower than those of the model group from the second week after administration (P<0.01). The fur scores of the JNW medium-dose group were significantly lower than those of the model group at the second week after administration (P<0.05), and the fur scores of the JNW low-dose group were significantly lower than those of the model group at the third and fourth weeks after administration (P<0.01). The fur scores of the JNW low-dose group were significantly lower than those of the model group at the fourth week after administration (P<0.05).

[0178] Effect of JNW on sucrose preference in CUMS model mice

[0179] Rodents show a strong preference for energy-rich foods such as sucrose. Sucrose has a rewarding nature and can stimulate the reward circuit in the rodent brain, producing a sense of pleasure. For depression model rodents, their preference for sugar water may be weakened, showing depression-like behaviors of anhedonia. The experimental results show ( Figure 3 ), after the mice were exposed to CUMS, the sucrose preference gradually decreased, and from the 2nd week after modeling, there was a very significant difference compared with the normal group mice (P<0.01). After giving different doses of JNW and fluoxetine, the sucrose preference of the model mice improved to varying degrees. Since the 3rd week of modeling, the fluoxetine group showed significant improvement compared with the model group, among which, there was a significant improvement compared with the model group at the 3rd week of modeling (P<0.05), and there was a very significant improvement compared with the model group at the 4th and 5th weeks of modeling (P<0.01). The JNW high-dose group showed a significant improvement compared with the model group at the 4th week (P<0.05), and there was a very significant improvement compared with the model group at the 5th week of modeling (P<0.01).

[0180] 3.4 Effect of JNW on the Tail Suspension Immobility Time of CUMS Model Mice

[0181] When mice are suspended upside down, they will struggle to get out of the predicament. If they still cannot get out of the predicament after trying hard, they will become intermittently immobile, showing a state of "behavioral despair". This state of "behavioral despair" is similar to depression. The level of "behavioral despair" of animals can be measured by the time they remain immobile while their tails are suspended. The results of the experiment found that ( Figure 4 ), after 6 weeks of CUMS stimulation, the immobility time of the tail suspension of the model group mice was significantly longer than that of the normal group, with a very significant difference (P<0.01). After administration of JNW, the immobility time of the tail suspension of mice decreased in a dose-dependent manner, among which the immobility time of the tail suspension of the medium-dose group was significantly reduced compared with the model group (P<0.05), and the immobility time of the tail suspension of the high-dose group was very significantly reduced compared with the model group (P<0.01).

[0182] Effect of 3.5JNW on the immobility time of forced swimming in CUMS model mice

[0183] Similar to the tail suspension test, in the forced swim test, when the animal realizes that it cannot escape the predicament, it will adopt an immobile posture and make only minimal efforts to keep its head above the water, showing a state of "behavioral despair". The level of "behavioral despair" of the animal can be measured by the immobility time of the forced swim. Figure 5It can be seen that after 6 weeks of CUMS stimulation, the immobility time of forced swimming in the model group was significantly longer than that in the normal group, with a very significant difference (P<0.01). After administration of JNW, the immobility time of forced swimming in the mice decreased in a dose-dependent manner, among which the immobility time of forced swimming in the medium-dose group and the high-dose group was significantly lower than that in the model group (P<0.05).

[0184] 3.6 Effect of JNW on the open field test behavior of CUMS model mice

[0185] Rodents often exhibit a "mural tropism" behavior characterized by avoidance of bright open spaces. The open field test uses this fear response to assess depression / anxiety related behaviors. Animals with less depression / anxiety are more likely to explore the center of the open field, while animals with more depression / anxiety reduce movement and exploration and tend to stay closer to the walls of the open field. Figure 6 ), the number of times the mice in the CUMS model group entered the central area of ​​the open field, the time they stayed in the central area, and the distance they moved were significantly lower than those in the normal group (P<0.01), showing a higher state of depression / anxiety. After administration of different doses of JNW and fluoxetine, these behaviors were significantly improved. The number of times the mice in each JNW group entered the central area of ​​the open field, the time they stayed in the central area, and the distance they moved increased in a dose-dependent manner, among which the number of times the high-dose group entered the central area of ​​the open field, the time they stayed in the central area, and the distance they moved were significantly increased compared with the model group (P<0.05). The number of times the mice in the fluoxetine group entered the central area of ​​the open field, the time they stayed in the central area, and the distance they moved were significantly increased compared with the model group (P<0.01).

[0186] Effects of 3.7JNW on neurotransmitters in CUMS model mice

[0187] The monoamine neurotransmitter hypothesis holds that the decrease in the levels of monoamine neurotransmitters such as 5-HT and NE is an important mechanism for the occurrence of depression. Antidepressant drugs such as selective serotonin (5-HT) reuptake inhibitors and monoamine oxidase inhibitors can increase the concentration of intersynaptic monoamine neurotransmitters, thereby improving depressive symptoms. This study found that ( Figure 7), the concentrations of 5-HT and NE in the hippocampus and serum of the CUMS model mice were significantly lower than those of the normal mice, with extremely significant differences (P<0.01). Fluoxetine, as a selective 5-hydroxytryptamine (5-HT) reuptake inhibitor, can significantly increase the concentration of 5-HT in the hippocampus and serum of CUMS model mice (P<0.05), but has no significant effect on NE. The concentrations of 5-HT in the hippocampus and serum of each dose group of JNW increased in a dose-dependent manner, among which the concentrations of 5-HT in the hippocampus and serum of the medium dose group were significantly different from those of the model group (P<0.05), and the concentrations of 5-HT in the hippocampus and serum of the high dose group were extremely significantly different from those of the model group (P<0.01). In addition, JNW had a certain effect on increasing the NE concentration in hippocampal tissue and serum. The NE concentration in hippocampal tissue of the high-dose group was significantly different from that in the model group (P<0.05), the serum NE concentration was extremely significantly different from that in the model group (P<0.01), and the serum NE concentration in the medium-dose group was significantly different from that in the model group (P<0.05).

[0188] Effects of 3.8JNW on the HPA axis in CUMS model mice

[0189] The hypothalamic-pituitary-adrenal axis (HPA) is an important part of the neuroendocrine system, connecting the central nervous system and the hormone system, and participating in the regulation of stress response. Abnormal activation of the HPA axis caused by continuous stress may be an important mechanism for the occurrence and development of depression. The results of the experiment showed that ( Figure 8 ), the levels of hypothalamic CRH and serum ACTH and CORT in CUMS model mice were significantly higher than those in normal mice, with extremely significant differences (P<0.01). Different doses of JNW and fluoxetine had a certain effect on reducing the levels of hypothalamic CRH and serum ACTH and CORT in CUMS model mice. Among them, the levels of hypothalamic CRH and serum ACTH and CORT in the high-dose JNW group were significantly lower than those in the model group (P<0.05); the levels of hypothalamic CRH and serum ACTH in the fluoxetine group were extremely significantly lower than those in the model group (P<0.01), and the CORT level was significantly lower than that in the model group (P<0.05).

[0190] Effect of 3.9JNW on the inflammatory response of CUMS model mice

[0191] 3.9.1 Effect of JNW on cytokine secretion in CUMS model mice

[0192] Abnormal activation of the immune system may be one of the important causes of depression. Long-term or excessive activity of inflammatory factors can interfere with the normal function of neurons, including the synthesis, release, reuptake and transmission of neurotransmitters, thereby affecting mood and cognitive function. This study found that the three cytokines IL-1β, IL-6 and TNF-α related to depression in the serum and hippocampal tissue of the CUMS model were significantly higher than those in the control group ( Fig. 9 ), the levels of IL-1β, IL-6, and TNF-α in the serum and hippocampal tissues of CUMS model mice were significantly increased compared with those in the normal group (P<0.01). The levels of IL-1β, IL-6, and TNF-α in the serum and hippocampal tissues of mice in the JNW groups and fluoxetine groups were reduced to a certain extent compared with those in the model group. Among them: in serum, the TNF-α content in the JNW low-dose group was significantly different from that in the model group (P<0.05), the IL-1β content in the JNW medium-dose group was significantly different from that in the model group (P<0.05), the IL-6 and TNF-α contents were extremely significantly different from those in the model group (P<0.01), and the above three cytokines in the JNW high-dose group were extremely significantly different from those in the model group (P<0.01); in hippocampal tissue, the IL-1β, IL-6 and TNF-α contents in the JNW medium-dose group were significantly different from those in the model group (P<0.05), and the above three cytokines in the JNW high-dose group were extremely significantly different from those in the model group (P<0.01).

[0193] Further study of the mRNA expression levels of three cytokines, IL-1β, IL-6, and TNF-α, in hippocampal tissue revealed that ( Fig.10 ), corresponding to the IL-1β, IL-6, and TNF-α protein expression levels in hippocampal tissue, the IL-1β, IL-6, and TNF-α mRNA expression levels in CUMS model mice were significantly increased compared with normal mice, and all had extremely significant differences (P<0.01). JNW can dose-dependently downregulate the mRNA expression of IL-1β, IL-6, and TNF-α in CUMS mice. Compared with the model group, the IL-1β mRNA expression level in the JNW medium-dose group was significantly reduced (P<0.05), while the IL-1β mRNA expression level in the JNW high-dose group was extremely significantly reduced (P<0.01). For IL-6 mRNA expression, the expression level in the JNW high-dose group was extremely significantly reduced compared with the model group (P<0.01). The TNF-α mRNA levels in the JNW low, medium, and high-dose groups were significantly downregulated compared with the model group, and there were extremely significant differences compared with the model group (P<0.01).

[0194] 3.9.2 Effect of JNW on NF-κB / NLRP3 signaling pathway in CUMS model mice

[0195] NLRP3 inflammasome and depression are intracellular multiprotein complexes that participate in a variety of innate immune processes related to infection, inflammation, and autoimmunity. After recognizing their respective ligands, multiple receptors such as glucocorticoid receptors and P2X7 receptors can activate the NF-κB pathway, thereby promoting the formation and activation of NLRP3 inflammasomes, further activating Caspase-1, and promoting the secretion of cytokines such as IL-1β, causing damage to neurons and neural circuits, and then inducing depressive symptoms. Therefore, the NF-κB / NLRP3 signaling pathway plays a very important role in the occurrence and development of depression.

[0196] This study found that Fig.11 ), the expression level of NLPR3 mRNA in the hippocampus of CUMS model mice was significantly upregulated compared with that in normal mice, with a very significant difference (P<0.01), but the expression level of Caspase-1 mRNA did not change significantly (P>0.05). The expression of NLPR3 mRNA in the hippocampus of mice in each dose group of JNW and fluoxetine group was significantly decreased compared with that in the model group (P<0.01), but there was no significant effect on the expression level of Caspase-1 mRNA (P>0.05).

[0197] The study on the expression of key proteins in the NF-κB / NLRP3 signaling pathway found that ( Fig.13 ), the NF-κB protein level in the hippocampus of CUMS model mice was not significantly different from that in the normal group (P>0.05), but the IκBα protein level was significantly decreased (P<0.01), while the phosphorylated IκBα level was significantly increased (P<0.01). The IκBα protein in the hippocampus of the JNW high-dose group was significantly increased compared with the model group, with statistical differences (P<0.01), while the phosphorylated IκBα levels in the JNW medium-dose and high-dose groups were significantly decreased compared with the model group (P<0.01). At the same time, Fig.12 As shown in the data, the expression of NLPR3 and Caspase-1 proteins in the hippocampus of CUMS model mice was significantly upregulated compared with that in the normal group (P<0.01). After JNW intervention, this trend was significantly restored. The expression of NLPR3 in the hippocampus of the high-dose JNW group was significantly lower than that in the model group (P<0.01), the expression of Caspase-1 in the hippocampus of the low-dose JNW group was significantly lower than that in the model group (P<0.05), and the expression of Caspase-1 in the hippocampus of the medium-dose and high-dose JNW groups was significantly lower than that in the model group (P<0.05).

[0198] 3.10 Effect of JNW on the expression of BDNF and TrκB in CUMS model mice

[0199] BDNF has the function of promoting synaptic plasticity and maintaining neuronal survival. TrκB is a specific receptor for BDNF. After BDNF binds to the specific receptor TrκB, it stimulates different downstream signal pathways and transmits the signal to the cell nucleus, thereby exerting an antidepressant effect. Fig.14 , Fig.15 The results showed that after CUMS stimulation, although the BDNF mRNA expression level of mice did not change significantly, the TrκB mRNA expression level was significantly reduced, which was significantly different from the normal group (P<0.05). JNW in each dose group increased the BDNF mRNA expression of CUMS mice to a certain extent, but there was no significant difference compared with the model group; JNW could dose-dependently upregulate the TrκB mRNA expression of CUMS mice, among which the JNW medium dose group had a significant difference compared with the model group (P<0.05), and the JNW high dose group had a very significant difference compared with the model group (P<0.01).

[0200] Further research on the expression levels of BDNF and TrκB proteins revealed that ( Fig.16 ), the expression levels of BDNF and TrκB proteins in the hippocampus of CUMS model mice were significantly lower than those in the normal group (P<0.05), but JNW could increase their expression in a dose-dependent manner. The expression levels of BDNF protein in the medium-dose and high-dose JNW groups were significantly different from those in the model group (P<0.05), and the expression level of TrκB protein was extremely significantly different from that in the model group (P<0.01).

[0201] 3.11 Effect of JNW on the expression of Bcl-2 and Bax in CUMS model mice

[0202] Bcl-2 and Bax are both members of the Bcl-2 family, of which Bcl-2 is an anti-apoptotic protein, while Bax is a pro-apoptotic protein. Bax can be translocated to the mitochondrial membrane under the stimulation of death signals, forming homodimers or multimers, and forming permeability transition pores on the mitochondrial membrane, releasing pro-apoptotic factors such as cytochrome C; while Bcl-2 can form heterodimers with Bax, inhibiting the translocation and dimerization of Bax, closing the permeability transition pore, and inhibiting apoptosis. Long-term chronic stimulation can lead to apoptosis of hippocampal neurons and play an important role in the occurrence and development of depression.

[0203] This study found that Figures 17 to 19As shown in the results, compared with normal mice, the expression of Bcl-2 mRNA in CUMS model mice was significantly reduced (P<0.01), while the expression of Bax mRNA was significantly increased (P<0.01). JNW had a significant improvement effect on this change. The expression of Bcl-2 mRNA in the three dose groups of JNW was significantly increased compared with the model group (P<0.01), while the expression of Bax mRNA was significantly decreased (P<0.01). Consistent with the mRNA expression level, the expression of Bcl-2 protein in CUMS model mice was significantly reduced compared with normal mice (P<0.01), while the expression of Bax protein was significantly increased compared with normal mice (P<0.01). The expression of Bcl-2 protein in the three dose groups of JNW was significantly increased compared with the model group (P<0.01), while the expression of Bax protein was significantly decreased (P<0.01).

[0204] In summary, JNW can significantly improve the depressive-like symptoms of CUMS depression model mice, improve the fur condition of mice, restore sucrose preference, increase the number, time and moving distance of entering the central area in the open field test, and reduce the immobility time in the tail suspension test and forced swimming test, suggesting that it can be used as a Chinese medicine for strengthening the brain, improving intelligence, nourishing the heart and calming the nerves, and has good application prospects in the treatment of depression. JNW can reduce the levels of 5-HT and NE in the hippocampus and serum of model mice, reduce the levels of CRH in the hypothalamus and ACTH and CORT in serum, reduce the production of inflammatory cytokines IL-1β, IL-6, and TNF-α, and inhibit IκBα phosphorylation, reduce IκBα degradation, inhibit the assembly and activation of NLRP3 inflammasome mediated by the NF-κB pathway, inhibit Caspase-1 maturation, reduce IL-1β secretion, increase the expression of BDNF and TrkB in hippocampal tissue, reduce the expression of Bax in hippocampal tissue, and increase the expression of Bcl-2, indicating that its antidepressant mechanism involves increasing monoamine neurotransmitters, reducing excessive activation of the HPA axis, alleviating neuroinflammation, increasing neurotrophic factors, and inhibiting neuronal apoptosis.

[0205] It is worth noting that the pathological mechanism of depression is very complex. The previous "monoamine neurotransmitter hypothesis", "HPA axis hypothesis", "neuroinflammation hypothesis", "neurotrophic factor hypothesis", etc., are difficult to independently explain the pathogenesis of depression. More and more studies believe that depression may be a systemic disease caused by the combined action of multiple pathological factors such as neurotransmitters, hormones, neuroinflammatory factors, neurotrophic factors, etc. caused by sustained stress, and involves numerous targets and signal pathways. As a treatment method with multiple components, multiple targets, and multiple pathways, traditional Chinese medicine has outstanding advantages in treating systemic diseases such as depression. This study shows that JNW can improve the symptoms of depression from multiple mechanisms, showing a good prospect for clinical application. Further in-depth research on it, such as the use of multi-omics and other research methods to clarify the effective ingredients and their targets and mechanisms of action, is of great significance for its clinical promotion and new drug development.

[0206] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. The use of Jiannao Pills in the preparation of a drug for treating depression, characterized in that: The brain-strengthening pill contains 20-35 parts of angelica sinensis, 10-15 parts of rhizoma dahliae, 20-27 parts of cistanche deserticola, 10-15 parts of dragon's tooth, 20-27 parts of yam, 10-15 parts of amber, 15-21 parts of schisandra chinensis, 5-8 parts of gastrodia elata, 4-6 parts of cypress seed, 5-8 parts of salvia miltiorrhiza, 15-21 parts of alpinia oxyphylla, 5-8 parts of ginseng, 10-15 parts of polygala tenuifolia, 5-8 parts of chrysanthemum, 10-15 parts of calamus, 7-11 parts of ochre, 10-15 parts of rhizoma arisaematis, 40-55 parts of spiny jujube seed and 20-27 parts of wolfberry.

2. The use of the Jiannao Pills according to claim 1 in the preparation of a drug for treating depression, characterized in that: The formula of Jiannao Wan is 25 parts of Angelica sinensis, 10 parts of Radix Gastrodiae, 20 parts of Cistanche deserticola, 10 parts of Dragon's Tooth, 20 parts of Dioscorea opposita, 10 parts of Amber, 15 parts of Schisandra chinensis, 5 parts of Gastrodia elata, 4 parts of Platycladus orientalis, 5 parts of Salvia miltiorrhiza, 15 parts of Alpinia oxyphylla, 5 parts of Ginseng, 10 parts of Polygala tenuifolia, 5 parts of Chrysanthemum, 10 parts of Acorus calamus, 7.5 parts of Ochre, 10 parts of Arisaema ciliata, 40 parts of Semen Ziziphi spinulosae, and 20 parts of Lycium barbarum.

3. The use of the Jiannao Pills according to claim 1 in the preparation of a drug for treating depression, characterized in that: The invention provides Jiannao Pills as a drug for treating depression with a dosage of 0.03 g / kg to 0.15 g / kg.

4. The use of the Jiannao Pills according to claim 1 in preparing a drug for treating depression, characterized in that: The invention comprises the use of Jiannao Pills in preparing medicines for increasing the concentration of 5-HT and NE in hippocampal tissue and serum.

5. The use of the brain-strengthening pills according to claim 1 in preparing a drug for treating depression, characterized in that: The invention comprises the use of Jiannao Pills in preparing a medicine for reducing the levels of hypothalamic CRH and serum ACTH and CORT.

6. The use of the Jiannao Pills according to claim 1 in preparing a drug for treating depression, characterized in that: The invention comprises the use of Jiannao Pills in preparing a medicine for reducing the contents of IL-1β, IL-6 and TNF-α in serum and hippocampal tissue.

7. Use of the brain-strengthening pills according to claim 1 in preparing a drug for treating depression, characterized in that: The invention comprises the use of Jiannao Pills in preparing a medicine for reducing the expression of NLPR3 mRNA in hippocampal tissue.

8. The use of the Jiannao Pills according to claim 1 in preparing a drug for treating depression, characterized in that: The invention comprises the use of Jiannao Pills in preparing a medicine for restoring the normal expression of NLPR3 and Caspase-1 proteins in hippocampal tissue.

9. The use of the Jiannao Pills according to claim 1 in preparing a drug for treating depression, characterized in that: The invention comprises the use of Jiannao Pills in preparing a medicine for up-regulating the expression of BDNF and TrκB.

10. The use of the Jiannao Pills according to claim 1 in preparing a drug for treating depression, characterized in that: The invention comprises the use of Jiannao Pills in preparing a medicine for up-regulating Bcl-2 mRNA expression and reducing Bax mRNA expression.

Images