Traditional Chinese medicine composition as well as preparation, pharmaceutical composition and application thereof

Abstract

The invention belongs to the technical field of traditional Chinese medicine, and particularly relates to a traditional Chinese medicine composition and a preparation, a pharmaceutical composition and application thereof. Aiming at qi and blood deficiency, heart and spleen injury and phlegm and blood stasis internal resistance caused by depression which is not healed for a long time or body weakness, the prescription is prepared from the following components in parts by weight: 15-30 parts of astragalus membranaceus, 3-9 parts of ginseng, 9-15 parts of roasted rhizoma atractylodis macrocephalae, 9-15 parts of angelica sinensis, 12-20 parts of poria with hostwood, 6-12 parts of longan aril, 6-12 parts of turmeric and 3-9 parts of honey-fried licorice root by taking qi reinforcing, blood nourishing, spleen strengthening, heart calming, phlegm reducing and blood stasis removing as a core treatment method. According to the composition, the effects of reducing phlegm and removing stasis are enhanced by adding turmeric, the anti-depression effect and the anti-neuroinflammation effect are remarkably enhanced, the curative effect is equivalent to that of an NLRP3 specific inhibitor MCC950, the composition can be prepared into multiple dosage forms such as decoction and granules, clinical application is convenient and fast, a new effective scheme is provided for traditional Chinese medicine treatment of depression, and the composition has important clinical popularization value.

Description

Technical Field

[0001] This application belongs to the technical field of traditional Chinese medicine, and particularly relates to a traditional Chinese medicine composition, its preparation, a pharmaceutical composition and applications thereof. Background Art

[0003] At present, the first-line treatment of depression in modern medicine mainly relies on chemically synthesized drugs, such as selective serotonin reuptake inhibitors (SSRIs), serotonin and norepinephrine reuptake inhibitors (SNRIs), etc. These drugs exert their therapeutic effects by regulating the levels of monoamine neurotransmitters. However, this treatment strategy has many bottlenecks: Firstly, the clinical response rate is not ideal, and about one-third of patients belong to treatment-resistant depression; Secondly, the drugs take effect slowly, usually requiring continuous administration for 2 to 4 weeks to show efficacy, increasing the risk of dropout in the initial stage of treatment; Moreover, side effects are common, such as nausea, weight gain, sexual dysfunction, etc., affecting patient compliance; In addition, withdrawal symptoms and disease recurrence are likely to occur after stopping the drug after long-term use. These limitations highlight that the existing treatment strategy has relatively single targets and fails to comprehensively cover the complex pathophysiology of depression.

[0004] In the long-standing traditional Chinese medicine theory system, depression mostly belongs to the categories of "depressive syndrome", "hysterical psychosis", "plum-pit qi", "hypochondriacal neurasthenia", etc. Traditional treatment mostly starts from the liver, heart, and gallbladder, believing that its main pathogenesis is emotional discomfort and stagnation of liver qi. Therefore, the basic principle of treatment is mostly "soothing the liver and regulating qi to relieve depression", and representative prescriptions include Bupleurum Liver-Regulating Powder, Free and Easy Wanderer Powder, etc. This theoretical system has good effects on the initial disease and the "depression" of excess syndrome. However, clinical practice has found that for patients with a long-term course of disease or those with congenital deficiencies in constitution (such as qi deficiency and blood deficiency constitutions), the pathogenesis has undergone a fundamental change. Long-term emotional injury first damages the heart and spleen. Worry and overthinking consume heart blood; Sorrow injures the spleen, and the spleen fails to transport and transform properly, resulting in insufficient production of qi and blood. Thus, a deficiency state with the pathological basis of "deficiency of both the heart and spleen and insufficiency of qi and blood" is formed. Qi deficiency leads to weak propulsion and slow blood circulation, resulting in blood stasis; Spleen deficiency leads to failure of transportation and transformation, and internal retention of water and dampness, which accumulates into phlegm. This is the state of "deficiency phlegm" and "deficiency stasis" with deficiency causing excess and deficiency-excess complexity. For such complex syndromes, if the method of soothing the liver and regulating qi is still used, not only will the curative effect be不佳, but it may even加重 the deficiency of healthy qi due to the dispersing and耗气 nature of pungent drugs. Therefore, there are clear treatment blind spots and theoretical limitations in the existing traditional Chinese medicine treatment plans for depression of "deficiency syndrome" or "deficiency-excess complex syndrome".

[0005] In recent years, with the interdisciplinary integration of psychiatry and immunology, the "neuroinflammatory hypothesis of depression" has moved from the margins to the mainstream, providing a completely new perspective for understanding depression. Research shows that chronic stress, as a core trigger for depression, can activate microglia—innate immune cells in the central nervous system—leading to the continuous release of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1α (IL-1α), and interleukin-6 (IL-6), inducing a persistent low-grade inflammatory state in specific brain regions (especially the hippocampus and prefrontal cortex, which are closely related to emotion and cognition). Among these, the NLRP3 inflammasome, an intracellular multi-protein complex, is a key hub in initiating the inflammatory response. Activated NLRP3 inflammasomes can activate Caspase-1, thereby promoting the maturation and release of potent pro-inflammatory factors such as IL-1β and IL-18. These inflammatory factors not only directly damage neuronal function, but have also been shown to decrease synaptic plasticity, lose dendritic spines, and reduce the expression of synaptic-related proteins (such as SYP and PSD-95) by affecting the expression of brain-derived neurotrophic factor (BDNF), inhibiting neurogenesis, and causing synaptic pruning, ultimately manifesting as depressive-like behavior. This series of findings provides a highly convincing modern biological interpretation of the "phlegm and blood stasis obstructing the brain's collaterals" pathogenesis in traditional Chinese medicine's "deficiency phlegm and deficiency stasis" syndrome. However, despite the clear mechanism, there is currently a lack of clinically available therapeutic drugs that can safely and effectively penetrate the blood-brain barrier, precisely regulate this pathway, and simultaneously meet the body's overall regulatory needs.

[0006] In conclusion, neither modern medical chemical drugs nor traditional Chinese medicine classic liver-soothing formulas are sufficient to address the complex and protracted depression characterized by "deficiency of both heart and spleen, and insufficiency of qi and blood," coupled with "internal obstruction of phlegm and blood stasis" (i.e., neuroinflammation and synaptic damage). Therefore, based on a deep understanding of the essence of traditional Chinese medicine's theory of "strengthening the body's resistance and eliminating pathogenic factors," and drawing on the latest understanding of the inflammatory mechanisms of depression in modern neuroscience, creating an innovative Chinese herbal compound that can "tonify qi and blood, strengthen the spleen and calm the mind" to support the body's resistance, while simultaneously "resolving phlegm and removing blood stasis" to directly inhibit neuroinflammation and repair synaptic damage, not only has significant theoretical value but also contains urgent clinical needs and broad development prospects. Summary of the Invention

[0007] To address the aforementioned problems, this application proposes a traditional Chinese medicine composition based on the principles of invigorating qi and nourishing blood, strengthening the spleen and calming the mind, and resolving phlegm and removing blood stasis. This composition is specifically designed for chronic depression or those caused by inherent weakness, resulting in qi and blood deficiency, damage to both the heart and spleen, and internal obstruction of phlegm and blood stasis. The specific technical solution is as follows: In the first aspect, this application proposes a traditional Chinese medicine composition comprising the following raw materials in parts by weight: Astragalus membranaceus 15-30 parts, Ginseng 3-9 parts, stir-fried Atractylodes macrocephala 9-15 parts, Angelica sinensis 9-15 parts, Poria cocos 12-20 parts, Longan aril 6-12 parts, Curcuma longa 6-12 parts, and prepared Glycyrrhiza uralensis 3-9 parts.

[0008] Furthermore, it is composed of the following raw materials in parts by weight: Astragalus membranaceus 20 parts, Ginseng 6 parts, stir-fried Atractylodes macrocephala 12 parts, Angelica sinensis 12 parts, Poria cocos 15 parts, longan pulp 10 parts, turmeric 9 parts, and prepared licorice root 6 parts.

[0009] Secondly, this application proposes a traditional Chinese medicine preparation containing the aforementioned traditional Chinese medicine composition.

[0010] Furthermore, the formulation types of the traditional Chinese medicine preparations include tablets, capsules, granules, pills, decoctions, or powders.

[0011] Thirdly, this application proposes a pharmaceutical composition in which at least one of the traditional Chinese medicine compositions is used as an active pharmaceutical ingredient.

[0012] Furthermore, the pharmaceutical composition also contains pharmaceutically acceptable excipients, including one or more of fillers, binders, disintegrants, lubricants, and solubilizers.

[0013] Thirdly, this application proposes the use of the described traditional Chinese medicine composition, or the described traditional Chinese medicine preparation, or the described pharmaceutical composition in the preparation of a medicament for treating and / or preventing depression.

[0014] Furthermore, the application exerts its antidepressant effect by inhibiting the activation of the NLRP3 inflammasome in hippocampal microglia.

[0015] Furthermore, the inhibition of NLRP3 inflammasome activation in hippocampal microglia includes reducing the expression levels of NLRP3, ASC, Caspase-1, and IL-1β proteins.

[0016] Furthermore, the application exerts an antidepressant effect by increasing the expression of hippocampal synaptic proteins SYP and PSD-95, thereby increasing dendritic spine density and the number of dendritic branches, in order to protect synaptic structure and function.

[0017] Compared with the prior art, this application has the following advantages: This application, based on the core pathogenesis of depression—"deficiency of Qi and blood, damage to both the heart and spleen, and internal obstruction of phlegm and blood stasis"—innovatively incorporates the principles of resolving phlegm and removing blood stasis into the classic treatment methods of tonifying Qi and nourishing blood, strengthening the spleen and calming the mind, and optimizes the formula by adding turmeric. This formula precisely matches the pathological mechanisms of neuroendocrine-immune network disorder, persistent low-grade neuroinflammation, and impaired synaptic plasticity under the background of chronic stress in depression, achieving a precise connection between traditional Chinese medicine pathogenesis and modern pathological mechanisms, resulting in stronger therapeutic targeting. Molecular biology experiments further clarified that its mechanism of action is to inhibit the activation of the NLRP3 inflammasome pathway, exert anti-neuroinflammatory effects, and protect synaptic function. Its efficacy is comparable to that of the NLRP3 specific inhibitor MCC950, providing a solid scientific basis for clinical application.

[0018] Compared with the classic formula Gui Pi Tang, the traditional Chinese medicine composition proposed in this application overcomes the limitations of simply tonifying the heart and spleen in improving core symptoms of depression and inhibiting neuroinflammation, and endows the formula with a clear anti-NLRP3 inflammasome activation effect. Comparative experiments have confirmed that the formula with Astragalus membranaceus as the principal ingredient and an appropriate amount of Curcuma longa as the adjuvant has the best comprehensive efficacy in improving depressive behavior, protecting synapses and anti-inflammation, which is significantly better than the scheme with ginseng as the principal ingredient or reduced dose of Astragalus membranaceus, fully demonstrating the rationality and innovation of the formula.

[0019] The medicines in this application can be prepared into decoctions using conventional water decoction methods, or into various dosage forms such as granules, tablets, and capsules using modern pharmaceutical technologies such as dynamic circulation extraction and low-temperature concentration. The preparation process is mature and simple, facilitating industrial production. Different dosage forms can meet the needs of different patients in clinical practice, improving the convenience of medication and compliance, and have broad clinical applicability.

[0020] Other features and advantages of this application will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the application. The objectives and other advantages of this application may be realized and obtained by means of the structures pointed out in the description, claims and drawings. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1The behavioral test results of mice in each experimental group in this application embodiment are shown; where A is the result of the sucrose preference test, showing the sucrose preference rate of mice in each group; B is the result of the novelty inhibition feeding test, showing the feeding latency of mice in each group; C and D are the results of the tail suspension test and forced swimming test, respectively, showing the immobility time of mice in each group. All data are expressed as mean ± standard deviation (n=12). Compared with the normal group, ### P < 0.001; compared with the model group, P<0.001. (Note: The model group (CUMS) is a chronic, unpredictable, mild stress group, and MCC950 is a control group of NLRP3 inhibitors.) Figure 2 The results of the sucrose preference test (A) and forced swimming test (B) for each group of mice in the comparative group of this application are shown; all data are expressed as mean ± standard deviation (n=10). Compared with the full-group group of this application, P<0.05, P<0.01, P<0.001; Figure 3 The results of the effects of each experimental group on the expression of synapse-related proteins in mice are shown in the embodiments of this application; where A is the representative band of SYP and PSD-95 synapse-related proteins in hippocampal tissue detected by Western blotting, and B is the semi-quantitative statistical analysis results of SYP and PSD-95 protein expression; all data are expressed as mean ± standard deviation (n=3). Compared with the normal group, ### P < 0.001; compared with the model group, P<0.001; Figure 4 The results of the effects of each experimental group on NLRP3 inflammasome pathway proteins in the embodiments of this application are shown; where A is the representative band of key proteins (NLRP3, ASC, Caspase-1, IL-1β) of the NLRP3 inflammasome pathway in hippocampal tissue detected by Western blotting; B is the semi-quantitative statistical analysis results of the expression of each protein (NLRP3, ASC, Caspase-1, IL-1β). All data are expressed as mean ± standard deviation (n=3). Compared with the normal group, # P<0.05, ### P < 0.001; compared with the model group, P<0.05, P<0.001; MCC950 was the NLRP3 inhibitor control group; Figure 5The following figures illustrate the effects of quantitative real-time PCR on the expression of pro-inflammatory cytokine mRNA in the hippocampus of CUMS depressed mice in each experimental group in this application embodiment; where A, B, and C are respectively... Tnfα , Il1α and C1qα The relative mRNA expression levels. All data are expressed as mean ± standard deviation (n=3). Compared with the normal group, ### P < 0.001; compared with the model group, P<0.001; MCC950 was the NLRP3 inhibitor control group; Figure 6 The results of quantitative real-time PCR (qRT-PCR) detection of the effects of each comparative experimental group on the expression of hippocampal pro-inflammatory factor mRNA in CUMS depressed mice are shown in the embodiments of this application; where A and B are the results of qRT-PCR detection of hippocampal pro-inflammatory factors, respectively. Tnfα and Il1β The relative mRNA expression levels, CE, and other parameters were used to detect key components of the NLRP3 inflammasome by qRT-PCR. Nlrp3 , Asc and Caspase-1 The relative expression levels of mRNA. All data are expressed as mean ± standard deviation (n = 4). Compared with the full-scale group of this application, P<0.05, P<0.01, P<0.001; Figure 7 The following diagram illustrates the Golgi staining and quantitative analysis results of hippocampal tissue from mice in each experimental group in this application embodiment; where A is a representative image of hippocampal neuron dendritic branches displayed by Golgi staining (scale bar: 50 µM); B is the result of quantitative statistical analysis of dendritic branch complexity using Sholl analysis; C is a representative image of hippocampal neuron dendritic spines displayed by Golgi staining (scale bar: 10 µM); D is the quantitative statistical analysis of dendritic spine density; all data are expressed as mean ± standard deviation (n=3). Compared with the normal group, ### P < 0.001; compared with the model group, P<0.001; MCC950 was the NLRP3 inhibitor control group; Figure 8 The results of immunofluorescence double-label staining and quantitative analysis of mice in different experimental groups in this application are shown; where A is a representative image of the co-localization of microglia (Iba-1, red) and NLRP3 (green) in the CA1 region of the hippocampus, as shown by immunofluorescence double-label staining; the cell nuclei are labeled with DAPI (blue). Scale bar: 500 µM, 50 µM; B is Iba-1 + / NLRP3 +Quantitative analysis of the number of double-positive cells. All data are expressed as mean ± standard deviation (n=3). Compared with the normal group, ### P < 0.001; compared with the model group, P<0.001; MCC950 was the control group for NLRP3 inhibitors. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0024] In response to persistent depression or underlying physical weakness leading to deficiency of Qi and blood, damage to both the heart and spleen, and internal obstruction of phlegm and blood stasis, this application proposes a traditional Chinese medicine composition consisting of eight herbs: Astragalus membranaceus, Ginseng, stir-fried Atractylodes macrocephala, Angelica sinensis, Poria cocos, Longan aril, Curcuma longa, and prepared Glycyrrhiza uralensis. This composition aims to improve the core symptoms of depression in patients and, by regulating the neuroendocrine-immune network, inhibit chronic low-grade neuroinflammation in the hippocampus, and protect synaptic structure and function, thereby achieving a therapeutic effect that addresses both the symptoms and the root cause.

[0025] This formula is based on the classic prescription "Gui Pi Tang," adhering to the principle of "tonifying qi and nourishing blood, strengthening the spleen and calming the mind" to support the body's vital energy and consolidate its foundation. By adding turmeric, which invigorates blood and reduces inflammation, and astragalus and angelica, which tonify qi and invigorate blood, the core of the formula is "tonifying while promoting circulation, and promoting circulation without harming the body's vital energy." The entire formula works synergistically to tonify qi and nourish blood, strengthen the spleen and calm the mind, and resolve phlegm and remove blood stasis, achieving an organic unity of supporting the body's vital energy to eliminate pathogenic factors and eliminating pathogenic factors to restore the body's vital energy. This precisely addresses the complex pathogenesis of chronic depression, which is characterized by "deficiency mixed with excess."

[0026] The traditional Chinese medicine composition proposed in this application uses Astragalus membranaceus and ginseng as the principal herbs, stir-fried Atractylodes macrocephala and Angelica sinensis as the assistant herbs, Poria cocos, longan pulp and turmeric as adjuvant herbs, and prepared licorice root as the guiding herb. Specifically, it is composed of the following raw materials in parts by weight: Astragalus membranaceus 15-30 parts, ginseng 3-9 parts, stir-fried Atractylodes macrocephala 9-15 parts, Angelica sinensis 9-15 parts, Poria cocos 12-20 parts, longan pulp 6-12 parts, turmeric 6-12 parts, and prepared licorice root 3-9 parts.

[0027] Principal drug: Astragalus: Sweet in taste, slightly warm in nature, and enters the spleen and lung meridians. In this formula, it is used as the principal ingredient to greatly replenish the qi of the spleen and lungs, thus nourishing the source of qi and blood production. When qi is abundant, blood flows smoothly; when qi is sufficient, body fluids are distributed, preventing the formation of "deficient phlegm" and "deficient blood stasis" at their root. Modern research shows that it has significant immunomodulatory and anti-inflammatory effects.

[0028] Ginseng: Sweet and slightly bitter in taste, slightly warm in nature, and enters the spleen, lung, and heart meridians. It greatly replenishes vital energy, calms the mind and improves intelligence, assists astragalus in enhancing its qi-tonifying power, and can also calm the mind and relieve palpitations, targeting the core deficiency of qi and blood.

[0029] Minister's Medicine: Stir-fried Atractylodes macrocephala: Bitter and sweet in taste, warm in nature, and enters the spleen and stomach meridians. It strengthens the spleen and dries dampness, and works synergistically with Astragalus membranaceus and ginseng to invigorate the middle jiao (digestive system), promote the transformation and transportation of nutrients, and fundamentally eliminate the source of phlegm.

[0030] Angelica sinensis: sweet and pungent in taste, warm in nature, and enters the liver, heart, and spleen meridians. It nourishes and invigorates blood, making it an essential herb for nourishing blood. When combined with Astragalus membranaceus, it embodies the essence of the principle that "qi is the commander of blood, and blood is the mother of qi," resulting in vigorous qi and abundant blood, and sufficient qi and blood.

[0031] Adjuvant: Poria cocos: It has a sweet and bland taste, and a neutral nature. It enters the heart, spleen, and kidney meridians. Its calming and tranquilizing effects are superior to those of Poria cocos. It can also promote diuresis and eliminate dampness, assisting Atractylodes macrocephala in removing dampness and resolving phlegm, and directly improving mental symptoms such as palpitations, anxiety, and insomnia.

[0032] Longan pulp: sweet in taste, warm in nature, and enters the heart and spleen meridians. It nourishes heart blood, benefits spleen qi, calms the mind, and when combined with angelica to nourish heart blood, and with ginseng and astragalus to benefit spleen qi, it is specifically used to treat deficiency of both heart and spleen.

[0033] Turmeric: pungent and bitter in taste, warm in nature, and enters the spleen and liver meridians. It is the key innovative ingredient in this formula. It excels at breaking up blood stasis, promoting qi circulation, and relieving pain. Its active ingredient, curcumin, is a star molecule in modern anti-neuroinflammatory research, capable of penetrating the blood-brain barrier and directly targeting neuroinflammation caused by "deficiency and blood stasis," demonstrating its "dispelling pathogenic factors" effect.

[0034] Used medicine: Prepared licorice root: sweet in taste, neutral in nature, and enters the heart, lung, spleen, and stomach meridians. It has two functions: first, to harmonize the other herbs, ensuring that the formula is nourishing without causing stagnation and warming without causing dryness; second, to tonify qi and harmonize the middle jiao, assisting the principal herb in enhancing its tonifying effect.

[0035] Example 1 In this embodiment, the Peiyuan Ningshen Decoction consists of: Astragalus membranaceus 20g, Ginseng 6g, stir-fried Atractylodes macrocephala 12g, Angelica sinensis 12g, Poria cocos 15g, longan pulp 10g, Curcuma longa 9g, and prepared licorice root 6g.

[0036] The preparation method is as follows: Take the above eight medicinal slices and place them in a decoction container. First, add water to cover the herbs by about 3-5 cm and soak for 30 minutes. Bring to a boil over high heat, then simmer over low heat for 30 minutes. Filter the liquid. Second, add water to cover the herbs, bring to a boil over high heat, then simmer over low heat for 20 minutes. Filter the liquid. Combine the two decoctions and concentrate to an appropriate volume (e.g., 300-400 ml) to obtain the decoction (named Peiyuan Ningshen Decoction).

[0037] If preparing powders, tablets, capsules, granules, or pills, the combined decoction is extracted using dynamic circulation, low-temperature concentration, and spray drying techniques to produce a dry extract. Pharmaceutical excipients are then added, and the corresponding dosage form is prepared according to conventional pharmaceutical processes.

[0038] Example 2 Pharmacodynamic studies were conducted on the Peiyuan Ningshen Decoction prepared in Example 1: 2.1 Test drugs and reagents.

[0039] Peiyuan Ningshen Decoction: The Peiyuan Ningshen Decoction prepared in Example 1.

[0040] MCC950: A specific inhibitor of the NLRP3 inflammasome, purchased from Selleck and prepared with sterile saline.

[0041] Main antibodies: anti-SYP, anti-PSD-95, anti-NLRP3, anti-ASC, anti-Caspase-1, anti-IL-1β, anti-Iba-1, etc., purchased from Abcam or CST.

[0042] 2.2 Experimental animals, grouping, and model establishment.

[0043] Healthy SPF-grade male C57BL / 6J mice, weighing 22-25g and aged 6-8 weeks, were provided by Hangzhou Ziyuan Experimental Animal Technology Co., Ltd. [License No.: SCXK (Zhejiang) 2019-0004] and housed at the Experimental Animal Center of the Institute of Neurology, Anhui University of Traditional Chinese Medicine. All mice were acclimatized for one week under standard conditions (12 / 12-hour light / dark cycle, temperature 22℃, free access to food and water).

[0044] 2.2.1 Grouping of research on main mechanisms.

[0045] Mice were randomly divided into 4 groups (n=12): Normal group: Normal C57BL / 6J mice, without modeling, were given an equal volume of physiological saline by gavage.

[0046] Model group (CUMS): Chronic mild stress-induced depression model mice. Mice were acclimatized to the new experimental environment for two weeks before modeling. During the first week, two bottles of 1% sucrose solution were provided, and during the second week, this was replaced with one bottle of clean tap water. After acclimatization, mice were housed individually and given 2-3 randomly assigned low-intensity social and environmental stressors daily, with different stressors on consecutive days, for a total of 6 weeks. Specific stress methods are shown in Table 1.

[0047] Table 1 CUMS Model Preparation

[0048] Peiyuan Ningshen Decoction Group (hereinafter referred to as Peiyuan Group): CUMS model mice were administered Peiyuan Ningshen Decoction solution by gavage.

[0049] MCC950 group (MCC group): CUMS model mice were intraperitoneally injected with MCC950 at a dose of 10 mg / kg.

[0050] 2.2.2 Comparative experimental grouping.

[0051] Another group of CUMS model mice was randomly divided into 4 control groups (n=10): The whole formula group (Astragalus membranaceus as the principal herb): The formula is the same as that of Peiyuan Ningshen Decoction group (Astragalus membranaceus 20g as the principal herb).

[0052] Gui Pi Tang group: Refer to the classic composition and dosage of Gui Pi Tang in the Chinese Pharmacopoeia (Astragalus membranaceus 20g, Ginseng 6g, stir-fried Atractylodes macrocephala 12g, Angelica sinensis 12g, Poria cocos 15g, Longan aril 10g, prepared licorice root 6g, containing the original formula Polygala tenuifolia 6g, Ziziphus jujuba var. spinosa 12g, Aucklandia lappa 6g, excluding Curcuma longa).

[0053] The group without turmeric: Remove turmeric from the Peiyuan Ningshen Decoction group, and keep the other ingredients and dosages unchanged (i.e., the formula is: Astragalus 20g, Ginseng 6g, stir-fried Atractylodes macrocephala 12g, Angelica sinensis 12g, Poria cocos 15g, Longan pulp 10g, and prepared licorice root 6g).

[0054] The reduced dosage group of turmeric: the dosage of turmeric was reduced to 4g, while the dosage of the other herbs remained unchanged (i.e., the formula was: Astragalus membranaceus 20g, Ginseng 6g, stir-fried Atractylodes macrocephala 12g, Angelica sinensis 12g, Poria cocos 15g, longan pulp 10g, turmeric 4g, and prepared licorice root 6g).

[0055] All CUMS mice underwent a 6-week chronic unpredictable mild stress model. Starting from week 3 of modeling, each treatment group was administered the corresponding drug solution (equivalent dose of raw drug) daily by gavage, while the normal group and the model group were administered an equal volume of physiological saline by gavage. The intervention continued until the end of week 6.

[0056] 2.3 Behavioral tests.

[0057] A series of behavioral tests were conducted at the end of week 6 to assess the mice’s depressive-like behavior.

[0058] 2.3.1 Sucrose Preference Test (SPT): Used to assess anhedonia. Mice were trained to adapt to sucrose water before the experiment. During the actual experiment, after a 24-hour period of fasting but not food, each mouse was given one pre-weighed bottle of 1% sucrose water and one bottle of pure water. One hour later, these were removed and the mice were weighed again. Sucrose preference rate = (Sucrose water consumption / (Sucrose water consumption + Pure water consumption)) × 100%.

[0059] The results are as follows Figure 1As shown in Figure A, it can be seen that the sucrose preference rate of mice in the model group was significantly lower than that in the normal group (P<0.01). Compared with the model group, the sucrose preference rate of mice in both the Peiyuan group and the MCC950 group was significantly higher (P<0.01).

[0060] 2.3.2 Novelty Inhibition Feeding Test (NSF): Used to assess anxiety-like behavior. Mice that have been fasted for 24 hours are placed in the center of an unfamiliar open area with a small amount of food particles placed in the center. The latency period from placement to the start of feeding (feeding latency) is recorded. The total amount of food consumed within 5 minutes after the test is completed is measured.

[0061] The results are as follows Figure 1 As shown in Figure B, compared with the normal group, the feeding latency of mice in the model group was significantly prolonged (P<0.01). Compared with the model group, the feeding latency of mice in the Peiyuan group and the MCC950 group was significantly shortened (P<0.01), while the total food intake within 5 minutes was not significantly different, excluding the influence of appetite.

[0062] 2.3.3 Tail Suspension Test (TST) and Forced Swimming Test (FST): Used to assess behavioral despair. In the tail suspension test, the mouse's tail is fixed and it is suspended upside down in a box. The immobility time in the last 4 minutes within a 6-minute period is recorded. In the forced swimming test, the mouse is placed in a transparent cylindrical container (25 cm high, 10 cm in diameter), with a water depth of 10 cm and a water temperature of 23-25℃. The immobility time in the last 4 minutes within a 6-minute period is recorded.

[0063] The results are as follows Figure 1 As shown in C and D, compared with the normal group, the immobility time of mice in the model group was significantly prolonged in both the tail suspension and forced swimming tests (P<0.01). Compared with the model group, the immobility time of mice in both the Peiyuan group and the MCC950 group was significantly shortened (P<0.01).

[0064] The results of the sucrose preference test and forced swimming test for mice in each control group are as follows: Figure 2 As shown in Figures A and B, the full-formula group of this application showed significantly better effects on improving anhedonia and behavioral despair than the Guipi Decoction group, the group without turmeric, and the group with reduced turmeric dosage. P <0.05). Among them, although the Guipi Decoction group showed improvement compared to the model group, its effect was not as good as the whole formula group of this application, indicating that simply tonifying the heart and spleen has limitations in improving certain core behavioral symptoms.

[0065] 2.4 Sample collection and molecular biological detection.

[0066] After the behavioral tests were completed, the mice were euthanized, and the hippocampus was quickly dissected for subsequent molecular biological testing. 2.4.1 Western Blot; Total protein was extracted from the hippocampus, and after the concentration was determined, SDS-PAGE electrophoresis, membrane transfer, and blocking were performed. SYP, PSD-95, NLRP3, ASC, Caspase-1, IL-1β, and internal control GAPDH primary antibody were added, and the mixture was incubated overnight at 4°C. The next day, the mixture was incubated with HRP-labeled secondary antibody, and the membrane was developed by ECL chemiluminescence. The gray values ​​of the bands were analyzed using ImageJ software.

[0067] Effects on synapse-related protein expression: Western blot results showed that, compared with the normal group, the expression levels of SYP and PSD-95 proteins in the hippocampus of the model group mice were significantly downregulated (P<0.05, e.g., Figure 3 (A and B). Intervention with Peiyuan Ningshen Decoction significantly reversed this trend, resulting in a significant increase in the expression levels of SYP and PSD-95 proteins (P<0.05, e.g., A and B). Figure 3 (Groups A and B) have similar effects to the MCC950 group.

[0068] Effects on NLRP3 inflammasome pathway proteins: Western blot results showed that, compared with the normal group, the expression levels of key proteins in the NLRP3 inflammasome pathway, NLRP3, ASC, Caspase-1, and mature IL-1β, were significantly upregulated in the hippocampus of the model group mice (P<0.05). Figure 4 (A and B). Treatment with Peiyuan Ningshen Decoction significantly inhibited the expression of these proteins (P<0.05, e.g., A and B). Figure 4 (A and B in the middle).

[0069] 2.4.2 Real-time quantitative PCR (qRT-PCR); Total RNA was extracted from the hippocampus and reverse transcribed into cDNA; the cDNA was detected using the SYBR Green assay on a real-time quantitative PCR instrument. Tnfα , Il1α , C1qα The mRNA expression level was determined using GAPDH as an internal control, and 2... ΔΔ Ct method for calculating relative expression levels.

[0070] qRT-PCR results showed that, compared with the normal group, the hippocampus tissue of the model group mice contained pro-inflammatory factors. Tnfα , Il1α and C1qα The mRNA expression levels were significantly increased (P<0.01, e.g.) Figure 5 As shown in AC). The intervention with Peiyuan Ningshen Decoction significantly inhibited the overexpression of these pro-inflammatory factors (P<0.01, as shown in AC). Figure 5 As shown in AC), its effect is similar to that of the MCC950 group.

[0071] Comparative experiment ( Figure 6 The whole-group group reduced hippocampal pro-inflammatory factors (AE): Tnfα , Il1β ) mRNA expression and inhibition of key mRNAs of the NLRP3 inflammasome ( Nlrp3 , Asc , Caspase-1 The expression of the inflammatory markers showed a significant advantage, with the anti-inflammatory effect being significantly better than that of the Guipi Decoction group without turmeric (P<0.01); while the Guipi Decoction group showed no statistical difference from the model group in these inflammatory markers, or the improvement was much smaller than that of the whole formula group.

[0072] 2.4.3 Golgi staining; Fresh hippocampal tissue was collected and processed according to the instructions of the Golgi staining kit. After imaging the stained neurons, the dendritic spine density and dendritic branching complexity (using Sholl analysis) were reconstructed and quantitatively analyzed using Neurolucida software.

[0073] Golgi staining results showed that, compared with the normal group, the model group mice had significantly reduced dendritic spine density and decreased dendritic branching complexity in hippocampal neurons (Sholl analysis showed a reduced number of crossover points, P<0.01, as shown in the figure). Figure 7 In the middle AD group, the dendritic spine density and dendritic branching complexity of mice were significantly improved (P<0.01, as shown in the figure). Figure 7 (Chinese AD).

[0074] 2.4.4 Immunofluorescence staining; Frozen sections of mouse brain tissue were prepared. After antigen retrieval, double immunofluorescence staining was performed using anti-Iba-1 (microglial cell marker) and anti-NLRP3 antibodies, and the cell nuclei were counterstained with DAPI. The number of Iba-1 and NLRP3 double-positive cells in the CA1 region of the hippocampus was observed and counted under a confocal microscope.

[0075] Immunofluorescence double labeling results showed that, compared with the normal group, the model group mice had an increased number of Iba-1 positive microglia in the CA1 region of the hippocampus, which were in an activated state, and the number of cells co-localized with NLRP3 was significantly increased (P<0.01, e.g., ...). Figure 8 (A and B). After intervention with Peiyuan Ningshen Decoction, Iba-1 + / NLRP3 + The number of double-positive cells was significantly reduced (P<0.01, e.g.) Figure 8 (A and B) indicates that Peiyuan Ningshen Decoction can specifically inhibit the activation of NLRP3 inflammasomes in microglia.

[0076] The above experiments demonstrate that the Peiyuan Ningshen Decoction of this application can significantly improve depressive-like behavior in CUMS mice. Its mechanism of action involves inhibiting the activation of the NLRP3 inflammasome in microglia, thereby reducing downstream pro-inflammatory factors (…). Tnfα , Il1β ,Il1α , C1qa The release of SYP / PSD-95 ultimately protects the structure and function of hippocampal synapses (increasing SYP / PSD-95 expression and dendritic spine density and complexity), thereby exerting an antidepressant effect. The efficacy of this formula is comparable to that of the specific NLRP3 inhibitor MCC950, fully validating its scientific connotation of "resolving phlegm and removing blood stasis" (i.e., anti-neuroinflammatory) to "nourish the body and calm the mind" (i.e., improve depressive symptoms).

[0077] Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application.

Claims

1. A traditional Chinese medicine composition, characterized in that, It is composed of the following raw materials in parts by weight: Astragalus membranaceus 15-30 parts, Ginseng 3-9 parts, stir-fried Atractylodes macrocephala 9-15 parts, Angelica sinensis 9-15 parts, Poria cocos 12-20 parts, longan pulp 6-12 parts, turmeric 6-12 parts, and prepared licorice root 3-9 parts.

2. The traditional Chinese medicine composition according to claim 1, characterized in that, It is composed of the following raw materials in parts by weight: Astragalus membranaceus 20 parts, Ginseng 6 parts, stir-fried Atractylodes macrocephala 12 parts, Angelica sinensis 12 parts, Poria cocos 15 parts, longan pulp 10 parts, turmeric 9 parts, and prepared licorice root 6 parts.

3. A traditional Chinese medicine preparation, characterized in that, The traditional Chinese medicine composition contains the one described in claim 1 or 2.

4. The traditional Chinese medicine preparation according to claim 3, characterized in that, The types of traditional Chinese medicine preparations include tablets, capsules, granules, pills, decoctions, or powders.

5. A pharmaceutical composition, characterized in that, At least one of the active pharmaceutical ingredients is the traditional Chinese medicine composition as described in claim 1 or 2.

6. The pharmaceutical composition according to claim 5, characterized in that, The pharmaceutical composition further contains pharmaceutically acceptable excipients, including one or more of fillers, binders, disintegrants, lubricants, and solubilizers.

7. The use of the traditional Chinese medicine composition of claim 1 or 2, or the traditional Chinese medicine preparation of claim 3 or 4, or the pharmaceutical composition of claim 5 or 6 in the preparation of a medicament for treating and / or preventing depression.

8. The application according to claim 7, characterized in that, The application exerts its antidepressant effect by inhibiting the activation of the NLRP3 inflammasome in hippocampal microglia.

9. The application according to claim 7, characterized in that, The application inhibits the activation of the NLRP3 inflammasome in hippocampal microglia by reducing the expression levels of NLRP3, ASC, Caspase-1, and IL-1β proteins.

10. The application according to claim 7, characterized in that, The application exerts its antidepressant effect by increasing the expression of hippocampal synaptic proteins SYP and PSD-95, thereby increasing dendritic spine density and the number of dendritic branches, in order to protect synaptic structure and function.

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