Application of composition consisting of lily and rehmannia in preparation of medicine for preventing and / or treating post-stroke depression

Abstract

The invention provides application of a composition consisting of lily and rehmannia in preparation of a medicine for preventing and / or treating post-stroke depression, and particularly belongs to the technical field of traditional Chinese medicines. The invention discloses application of a composition consisting of lily and rehmannia in preparation of a medicine for preventing and / or treating post-stroke depression. The composition composed of the lily and the rehmannia has a treatment effect on post-stroke depression, the treatment effect is reflected in behavioral improvement, neuroprotection, nerve cell apoptosis inhibition, brain inflammation level reduction and neurotrophic pathway activation, and the ratio of the lily and rehmannia drug pairs is 1: 1, which is significantly better than the ratio of the lily and rehmannia drug pairs in 1: 0.5 and 1: 1.43.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, specifically relating to the application of a composition of lily and rehmannia in the preparation of a drug for the prevention and / or treatment of post-stroke depression. Background Technology

[0002] The current status of post-stroke depression (PSD): Post-stroke depression (PSD) is one of the most common and disabling neuropsychiatric complications after stroke, posing a significant challenge to patient prognosis. Epidemiological studies show that PSD usually occurs within 3 months after stroke and can last for several years, severely delaying the recovery of neurological function. PSD not only causes patients to experience persistent low mood, loss of interest, and cognitive impairment, but is also closely related to increased mortality, poor rehabilitation adherence, significantly reduced quality of life, and severe impairment of social function, placing a heavy care and economic burden on families and society.

[0003] Currently, the first-line treatment for PSD primarily involves antidepressants such as selective serotonin reuptake inhibitors (SSRIs), but their application has significant limitations. The side effects of SSRIs are considerable, commonly including gastrointestinal reactions (nausea, diarrhea), sexual dysfunction, and insomnia. Some SSRIs also pose a potential risk of interaction with anticoagulants, creating a medication hazard for stroke patients who require long-term antiplatelet therapy. Furthermore, abrupt discontinuation can trigger withdrawal symptoms. These limitations underscore the urgent need to develop novel treatment strategies based on different mechanisms of action, offering greater safety and proven efficacy.

[0004] Advantages of Traditional Chinese Medicine (TCM) in Treating Post-Stroke Depression (PSD): TCM possesses unique theoretical advantages and clinical value in treating post-stroke depression. TCM believes the core pathogenesis of PSD lies in "deficiency of the root and excess of the branch." "Deficiency of the root" primarily refers to liver and kidney yin deficiency and heart blood deficiency. Stroke, located in the brain, is closely related to the liver and kidneys; liver and kidney yin deficiency leads to depletion of essence and blood, resulting in malnourishment of the brain marrow. The heart governs the mind, and heart blood is the material basis for mental activity; insufficient heart blood leads to malnourishment of the mind, as stated in *Danxi Xinfa*: "Blood deficiency leads to a lack of nourishment for the spirit," resulting in low mood and lethargy. "Excess of the branch" manifests as the mutual binding of phlegm and blood stasis due to deficiency, and internal disturbance of deficiency fire. The pathological products of stroke—"wind, fire, phlegm, and blood stasis"—obstruct the brain orifices, disturb the mind, and exacerbate mental sluggishness and emotional disorders, forming a complex situation of mixed deficiency and excess.

[0005] Based on this holistic view of pathogenesis, the advantages of traditional Chinese medicine (TCM) in treating PSD lie in its "multi-target intervention" and "holistic regulation." Unlike Western medicine's single-target approach (such as selective inhibition of 5-HT reuptake), TCM herbal pairs work synergistically through multiple active ingredients on multiple pathological processes, including neuroendocrine, neuroinflammation, neurotrophic factors, and neurotransmitters, achieving comprehensive therapeutic effects. This integrative regulatory effect aligns with the complex pathophysiological mechanisms of PSD.

[0006] In addition, while improving the core symptoms of depression, traditional Chinese medicine can also significantly promote the recovery of neurological functions (such as motor and cognitive functions) after stroke, improve the quality of life, and has good overall safety with few side effects. When used in combination with Western medicine, it can reduce toxicity and enhance efficacy, providing another important treatment option for patients with PSD.

[0007] Traditional application of the lily and rehmannia herbal pair: This herbal pair originates from the *Synopsis of Prescriptions of the Golden Chamber*. The original text describes it as follows: seven lily bulbs (split) and one liter of rehmannia juice. Wash the lily bulbs with water and soak overnight. When white foam appears, discard the water. Then, add two liters of spring water and decoct until one liter remains. Remove the dregs, add the rehmannia juice, and decoct until one and a half liters remain. Divide into two warm doses. Do not take more if the illness is cured. Stools should be as black as lacquer. Modern pharmacological research has found that lily bulbs have sedative, antidepressant, and neuromodulatory effects; rehmannia has anti-inflammatory, antioxidant, and neuroprotective effects.

[0008] Although there are clinical reports on the pharmacological effects of lily and rehmannia, the differences in efficacy, molecular mechanisms, key targets, and signaling pathways of different ratios of lily and rehmannia on PSD are still unclear. The lack of systematic modern pharmacological research evidence severely limits their modernization and clinical application. Summary of the Invention

[0009] The purpose of this invention is to provide the use of a composition of lily and rehmannia in the preparation of a medicament for the prevention and / or treatment of post-stroke depression. The composition of this invention has the advantages of multi-target therapy and fewer side effects.

[0010] This invention provides the use of a composition of lily and rehmannia in the preparation of a medicament for the prevention and / or treatment of post-stroke depression; the composition is obtained by mixing lily aqueous extract and rehmannia aqueous extract in a volume ratio of 1:0.5, 1:1 or 1:1.43.

[0011] The present invention also provides the application of a composition of lily and rehmannia in the preparation of a drug for inhibiting neuroinflammation; the composition is obtained by mixing lily aqueous extract and rehmannia aqueous extract in a volume ratio of 1:0.5, 1:1 or 1:1.43.

[0012] Preferably, the inhibition of neuroinflammation includes inhibiting neuroinflammation by regulating the levels of pro-inflammatory factors TNF-α, IL-1β, and IL-6 and by regulating the expression of GFAP and Iba1 proteins.

[0013] The present invention also provides the application of a composition of lily and rehmannia in the preparation of a drug for inhibiting nerve cell apoptosis; the composition is obtained by mixing lily aqueous extract and rehmannia aqueous extract in a volume ratio of 1:0.5, 1:1 or 1:1.43.

[0014] The present invention also provides the application of a composition of lily and rehmannia in the preparation of a drug that promotes nerve repair; the composition is obtained by mixing lily aqueous extract and rehmannia aqueous extract in a volume ratio of 1:0.5, 1:1 or 1:1.43.

[0015] The present invention also provides the use of a composition of lily and rehmannia in the preparation of drugs that increase the expression levels of BDNF, TrkB, ERK, PI3K or Akt proteins; the composition is obtained by mixing lily aqueous extract and rehmannia aqueous extract in a volume ratio of 1:0.5, 1:1 or 1:1.43.

[0016] Preferably, the method for preparing the lily water extract includes the following steps: mixing lily with water for water extraction to obtain a first residue and a first water extract; mixing the first residue with water for water extraction to obtain a second residue and a second water extract; mixing the first water extract and the second water extract and concentrating to obtain the lily water extract. The preparation method of the Rehmannia glutinosa aqueous extract includes the following steps: mixing Rehmannia glutinosa with water for water extraction to obtain a first residue and a first aqueous extract; mixing the first residue with water for water extraction to obtain a second residue and a second aqueous extract; mixing the first aqueous extract and the second aqueous extract and concentrating to obtain the Rehmannia glutinosa aqueous extract.

[0017] Preferably, the concentration of the concentrated lily water extract is 0.5 g crude drug / ml.

[0018] Preferably, the concentration of the concentrated Rehmannia glutinosa aqueous extract is 1.5g crude drug / ml.

[0019] Preferably, the drug is an oral preparation, which includes tablets, capsules, granules, or oral liquids.

[0020] This invention provides the application of a composition of lily and rehmannia in the preparation of a medicament for the prevention and / or treatment of post-stroke depression. Through systematic research comparing the efficacy differences of different ratios of lily and rehmannia, this invention determines the optimal ratio of the lily and rehmannia combination, elucidates its scientific implications for treating PSD, and provides a solid theoretical basis and experimental data for effective drug development. It treats post-stroke depression from multiple targets, demonstrating significant advantages over first-line antidepressants. Specifically, this invention constructs an in vivo pharmacodynamic model, using middle cerebral artery occlusion (MCAO) combined with chronic mild unpredictable stress to establish a stable and reliable PSD rat model. Behavioral tests (sucrose preference test, forced swimming test, and spontaneous activity test) are used to evaluate the behavioral improvement effects of different ratios of Lilium brownii and Rehmannia glutinosa on PSD rats. This invention also investigates the neuroinflammatory (anti-inflammatory) mechanism, evaluating the anti-neuroinflammatory effects of different ratios of Lilium brownii and Rehmannia glutinosa on PSD rats by detecting the levels of inflammatory factors (TNF-α, IL-1β, IL-6), inflammation status (HE staining), and the expression and distribution of GFAP and Iba-1 proteins in the brain tissue of PSD rats. The experimental results showed that different ratios of Lilium and Rehmannia glutinosa had significant behavioral improvement effects on rats with post-stroke depression (PSD), manifested as relief of depressive-like behaviors and activity inhibition, with the 1:1 ratio of Lilium and Rehmannia glutinosa being more effective. Different ratios of Lilium and Rehmannia glutinosa also inhibited neuronal apoptosis to varying degrees in rats with PSD, improved the damage status of neurons in the hippocampus, and promoted a series of neuroprotective effects, including neuronal structural and functional repair, with the 1:1 ratio of Lilium and Rehmannia glutinosa being more effective. The drug pairs exhibited significant anti-neuroinflammatory effects on rats with post-stroke depression (PSD), manifested as systemic inflammation relief and improvement of local hippocampal pathology, with the 1:1 ratio of the Lilium and Rehmannia glutinosa drug pairs being superior. All different ratios of the Lilium and Rehmannia glutinosa drug pairs could activate key proteins in downstream MAPK / ERK and PI3K / Akt signaling pathways by promoting the upregulation of the expression of neurotrophic factor BDNF and its receptor TrkB in hippocampal tissue. Furthermore, the 1:1 ratio of the Lilium and Rehmannia glutinosa drug pairs was significantly better than the 1:0.5 and 1:1.43 ratios.

[0021] The beneficial effects of this invention are as follows: This invention provides the first systematic and in-depth experimental evidence of the effectiveness of Lilium and Rehmannia glutinosa in treating PSD through multiple targets (neuroinflammation, neurotrophic and repair).

[0022] The results of this invention can be used to clinically optimize the ratio of lily and rehmannia, and complement classical Chinese medicine theories. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 A flowchart illustrating the preparation method of Lilium arvense extract provided by this invention; Figure 2 The effect of different ratios of Lilium and Rehmannia glutinosa provided by the present invention on the sugar water preference rate of rats in each group is shown in the figure. Figure 3 The effect of different ratios of Lilium and Rehmannia glutinosa drug pairs provided by the present invention on the immobility time of rats in each group during forced swimming is shown in the figure. Figure 4 The effect of different ratios of Lilium and Rehmannia glutinosa provided by this invention on the total spontaneous activity distance of rats in each group is shown in the figure. Figure 5 The following graphs show the effects of different ratios of Lilium and Rehmannia glutinosa (Lilium and Rehmannia) on the spontaneous activity trajectories of rats in each group after 3 weeks of administration, as provided in this invention. A represents the sham-operated group; B represents the PSD model group; C represents the positive control group (fluoxetine); D represents the high-dose Lilium and Rehmannia glutinosa (1:0.5); E represents the low-dose Lilium and Rehmannia glutinosa (1:0.5); F represents the high-dose Lilium and Rehmannia glutinosa (1:1.43); G represents the low-dose Lilium and Rehmannia glutinosa (1:1.43); H represents the high-dose Lilium and Rehmannia glutinosa (1:1); and I represents the low-dose Lilium and Rehmannia glutinosa (1:1). Figure 6 The following are the results of TTC staining of different ratios of Lilium and Rehmannia glutinosa drug pairs on cerebral infarction in rats after 3 weeks of administration, as provided in this invention; where A is the result of the sham-operated group; B is the result of the PSD model group; C is the result of the positive control group (fluoxetine); D is the result of the high-dose group of Lilium and Rehmannia glutinosa 1:0.5; E is the result of the low-dose group of Lilium and Rehmannia glutinosa 1:0.5; F is the result of the high-dose group of Lilium and Rehmannia glutinosa 1:1.43; G is the result of the low-dose group of Lilium and Rehmannia glutinosa 1:1.43; H is the result of the high-dose group of Lilium and Rehmannia glutinosa 1:1; and I is the result of the low-dose group of Lilium and Rehmannia glutinosa 1:1. Figure 7The following figures illustrate the effects of different ratios of Lilium and Rehmannia glutinosa drug pairs provided by this invention on the morphology of hippocampal neurons in rats after 3 weeks of administration (Nissl staining). A represents the sham-operated group; B represents the PSD model group; C represents the positive control group (fluoxetine); D represents the high-dose Lilium and Rehmannia glutinosa 1:0.5 group; E represents the low-dose Lilium and Rehmannia glutinosa 1:0.5 group; F represents the high-dose Lilium and Rehmannia glutinosa 1:1.43 group; G represents the low-dose Lilium and Rehmannia glutinosa 1:1.43 group; H represents the high-dose Lilium and Rehmannia glutinosa 1:1 group; and I represents the low-dose Lilium and Rehmannia glutinosa 1:1 group. Figure 8 The following figures illustrate the effects of different ratios of Lilium and Rehmannia glutinosa drug pairs provided by this invention on the survival and apoptosis of hippocampal neurons in rats after 3 weeks of administration (TUNEL staining). A represents the sham-operated group; B represents the PSD model group; C represents the positive control group (fluoxetine); D represents the high-dose Lilium and Rehmannia glutinosa 1:0.5 group; E represents the low-dose Lilium and Rehmannia glutinosa 1:0.5 group; F represents the high-dose Lilium and Rehmannia glutinosa 1:1.43 group; G represents the low-dose Lilium and Rehmannia glutinosa 1:1.43 group; H represents the high-dose Lilium and Rehmannia glutinosa 1:1 group; and I represents the low-dose Lilium and Rehmannia glutinosa 1:1 group. Figure 9 The effect of different ratios of Lilium and Rehmannia glutinosa drug pairs provided by the present invention on the serum levels of inflammatory factors IL-6, IL-1β and TNF-α in rats of each group after 3 weeks of administration; Figure 10 The following are the results (HE staining) of the effects of different ratios of Lilium and Rehmannia glutinosa on brain tissue inflammation in rats after 3 weeks of administration, as provided by this invention. A represents the sham-operated group; B represents the PSD model group; C represents the positive control group (fluoxetine); D represents the high-dose Lilium and Rehmannia glutinosa 1:0.5 group; E represents the low-dose Lilium and Rehmannia glutinosa 1:0.5 group; F represents the high-dose Lilium and Rehmannia glutinosa 1:1.43 group; G represents the low-dose Lilium and Rehmannia glutinosa 1:1.43 group; H represents the high-dose Lilium and Rehmannia glutinosa 1:1 group; and I represents the low-dose Lilium and Rehmannia glutinosa 1:1 group. Figure 11 The Western blot results show the effects of different ratios of Lilium and Rehmannia glutinosa drug pairs provided by this invention on the expression of GFAP and Iba-1 proteins in the hippocampus of rats in each group after 3 weeks of administration. Figure 12 Provided by the present invention Figure 11 Quantitative results graph; Figure 13The following figures illustrate the effects (immunohistochemical) of different ratios of Lilium and Rehmannia glutinosa drug pairs administered for 3 weeks on the distribution of astrocytes (GFAP) and microglia (Iba1) in the hippocampus of rats in each group. A represents the sham-operated group; B represents the PSD model group; C represents the positive control group (fluoxetine); D represents the high-dose Lilium and Rehmannia glutinosa 1:0.5 group; E represents the low-dose Lilium and Rehmannia glutinosa 1:0.5 group; F represents the high-dose Lilium and Rehmannia glutinosa 1:1.43 group; G represents the low-dose Lilium and Rehmannia glutinosa 1:1.43 group; H represents the high-dose Lilium and Rehmannia glutinosa 1:1 group; and I represents the low-dose Lilium and Rehmannia glutinosa 1:1 group. Figure 14 The effect of different ratios of Lilium and Rehmannia glutinosa drug pairs provided by the present invention on the expression of BDNF and TrkB proteins in the hippocampus of rats in each group after 3 weeks of administration (Western blot results); Figure 15 Provided by the present invention Figure 14 Quantitative results graph; Figure 16 The figure shows the effect of different ratios of Lilium and Rehmannia glutinosa drug pairs provided by the present invention on the expression of MAPK, ERK, PI3K and Akt proteins in the hippocampus of rats in each group after 3 weeks of administration. Figure 17 Provided by the present invention Figure 16 The quantitative results are shown in the figure. Detailed Implementation

[0025] This invention provides the use of a composition of lily and rehmannia in the preparation of a medicament for the prevention and / or treatment of post-stroke depression; the composition is obtained by mixing lily aqueous extract and rehmannia aqueous extract in a volume ratio of 1:0.5, 1:1, or 1:1.43. The composition of this invention has a therapeutic effect on post-stroke depression, manifested in behavioral improvement, neuroprotection, inhibition of neuronal apoptosis, reduction of brain inflammation levels, and activation of neurotrophic pathways. Furthermore, the 1:1 ratio of lily and rehmannia is significantly superior to the 1:0.5 and 1:1.43 ratios. In a specific embodiment, the preparation method of the lily water extract includes the following steps: mixing lily with water for water extraction to obtain a first residue and a first water extract; mixing the first residue with water for water extraction to obtain a second residue and a second water extract; mixing the first water extract and the second water extract, and concentrating to obtain the lily water extract. The preparation method of the rehmannia water extract includes the following steps: mixing rehmannia with water for water extraction to obtain a first residue and a first water extract; mixing the first residue with water for water extraction to obtain a second residue and a second water extract; mixing the first water extract and the second water extract, and concentrating to obtain the rehmannia water extract. In a specific embodiment, the water extraction method includes decoction or reflux extraction. In a specific embodiment, the concentration of the concentrated lily water extract is 0.5 g crude drug / ml. In a specific embodiment, the concentration of the concentrated rehmannia water extract is 1.5 g crude drug / ml. In a specific embodiment, the drug is an oral preparation, which includes tablets, capsules, granules, or oral liquid. The composition and preparation of the composition and the dosage form of the drug described below are the same and will not be repeated here.

[0026] This invention also provides the application of a composition of lily and rehmannia in the preparation of a drug for inhibiting neuroinflammation; the composition is obtained by mixing lily aqueous extract and rehmannia aqueous extract at a volume ratio of 1:0.5, 1:1, or 1:1.43. In a specific embodiment, the inhibition of neuroinflammation includes inhibiting neuroinflammation by regulating the levels of pro-inflammatory factors TNF-α, IL-1β, and IL-6, and by regulating the expression of GFAP and Iba1 proteins.

[0027] This invention also provides the application of a composition of lily and rehmannia in the preparation of a drug for inhibiting neuronal apoptosis; the composition is obtained by mixing lily aqueous extract and rehmannia aqueous extract at a volume ratio of 1:0.5, 1:1, or 1:1.43. The composition of this invention can inhibit neuronal apoptosis in post-stroke depression.

[0028] This invention also provides the application of a composition of lily and rehmannia in the preparation of a drug that promotes nerve repair; the composition is obtained by mixing lily aqueous extract and rehmannia aqueous extract at a volume ratio of 1:0.5, 1:1, or 1:1.43. The composition of this invention can promote nerve repair in post-stroke depression.

[0029] This invention also provides the application of a composition of lily and rehmannia in the preparation of drugs that increase the expression levels of BDNF, TrkB, ERK, PI3K, or Akt proteins; the composition is obtained by mixing lily aqueous extract and rehmannia aqueous extract at a volume ratio of 1:0.5, 1:1, or 1:1.43. The composition of this invention can promote the upregulation of the expression of neurotrophic factor BDNF and its receptor TrkB in hippocampal tissue of patients with post-stroke depression, activating key proteins in downstream MAPK / ERK and PI3K / Akt signaling pathways.

[0030] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, illustrates the application of a composition of lily and rehmannia provided by the present invention in the preparation of a medicament for the prevention and / or treatment of post-stroke depression, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0031] Example 1 A method for preparing a composition consisting of lily and rehmannia.

[0032] Materials: Electronic balance; induction cooker; round drum; purified water. Lily bulb (batch number: 240902, Bozhou Jingwan Chinese Medicine Pieces Factory), Rehmannia glutinosa (batch number: D2409167, Sichuan Xinhehua Chinese Medicine Pieces Co., Ltd.).

[0033] Lily bulb and Rehmannia glutinosa were extracted twice by reflux with purified water, each time for 1 hour. After reflux, the extracts were combined and concentrated by vacuum distillation to a concentration of 0.5 g crude drug / ml for the lily bulb aqueous extract and 1.5 g crude drug / ml for the Rehmannia glutinosa aqueous extract. They were stored at -20℃ for later use. Before administration, the lily bulb aqueous extract and Rehmannia glutinosa aqueous extract were mixed at volume ratios of 1:0.5, 1:1.43, and 1:1 to obtain compositions of lily bulb and Rehmannia glutinosa with different ratios.

[0034] Figure 1 This is a flowchart of a method for preparing a composition consisting of lily and rehmannia.

[0035] Example 2 Establishment of PSD rat model and administration regimens of different ratios of Lilium and Rehmannia glutinosa.

[0036] Materials: Portable multi-channel small animal anesthesia machine; isoflurane; restraint straps; MCAO suture plugs; surgical sutures; vascular clamps; stainless steel cylinders; swallowtail clips; penicillin (Nanyang Tianxing Agricultural Co., Ltd.); SD rats (Beijing Huafukang Biotechnology Co., Ltd.); fluoxetine (Patheon France).

[0037] method: ① Establishment of the PSD rat model First, a rat model of cerebral ischemia (MCAO) is constructed. After the model is established, a rat model of depression is then established. Once completed, the rat model of PSD is obtained.

[0038] MCAO model: Rats were anesthetized using a small animal anesthesia machine equipped with isoflurane and fixed in a supine position. The fur on the rat's neck was clipped, and after disinfection with alcohol, a longitudinal incision of about 2-3 cm was made about 0.5-1 cm above the line connecting the two forelimbs and 0.5 cm to the left of the midline of the neck. The tissue was dissected layer by layer to free the left common carotid artery, internal carotid artery, and external carotid artery. The proximal end of the common carotid artery was clamped with a vascular clamp, and a small incision was made below the bifurcation of the common carotid artery. The MCAO suture was inserted into the rat's internal carotid artery until resistance was encountered. The insertion depth was at least beyond the black mark line of the suture. The suture was tied and fixed, and the incision was closed in sequence. After the operation, the animal was placed in a cage with a heating blanket. After the rat was fully awake, it was transferred to a feeding box with clean bedding. Each rat was intraperitoneally injected with 80,000 U of penicillin for 3 consecutive days to prevent postoperative infection.

[0039] Indicators of model success: 1) After recovery from anesthesia, rats exhibited unilateral limb paralysis, unsteady standing, or circling to one side when their tails were lifted. 2) Neurological deficit assessment: Rats were assessed 24 hours after recovery from anesthesia using the Longa and Bederson 5-point scale. Rats scoring 1-3 points were selected for inclusion in the group. The neurological deficit assessment criteria were: "No neurological damage symptoms": 0 points; "Inability to fully extend the contralateral forepaw": 1 point; "Circling to the contralateral side": 2 points; "Tilting to the contralateral side": 3 points; "Inability to walk spontaneously, loss of consciousness": 4 points.

[0040] Three days after the MCAO model was established, depression modeling began. Rats were subjected to 2-3 stress stimuli daily (chronic mild unpredictable stress depression model) for 2 weeks. The stress stimuli were randomly selected and included tail clamping (2 min), hot water (45 degrees for 5 min), noise (3 h), ice water (4 degrees for 5 min), forced swimming (5 min), continuous restraint (1 h), fasting (24 h), water restriction (24 h), 45° inclined cage (24 h), moist bedding (24 h), day-night reversal (12 h light-dark alternation), and single-cage housing (continuous throughout the modeling period).

[0041] ②Lily and Rehmannia glutinosa drug pair dosing regimen This experiment consisted of 9 groups. The sham-operated group (purified water) consisted of rats without modeling, which underwent surgery only by opening and suturing. The remaining PSD model rats were randomly divided into 8 groups according to body weight and sex: PSD model group; positive control group (fluoxetine); high-dose Lilium brownii (1:0.5) group; low-dose Lilium brownii (1:0.5) group; high-dose Lilium brownii (1:1.43) group; low-dose Lilium brownii (1:1.43) group; high-dose Lilium brownii (1:1) group; and low-dose Lilium brownii (1:1) group. Each group contained 10 male rats.

[0042] According to the 2020 edition of the Chinese Pharmacopoeia: the clinical dosage of lily is 12g / 60kg; the clinical dosage of rehmannia is 15g / 60kg; the clinical dosage of lily and rehmannia is 27g / 60kg. The oral administration volume for rats is 10ml / kg. For adults weighing 60kg, the equivalent dose for rats is 2.78g of crude drug / kg.

[0043] The sham-operated group and the PSD model group were given purified water; the positive control group was given fluoxetine at a concentration of 1 mg / ml and a dosage of 5 mg / kg; Lily bulb rehmannia (1:0.5) at a concentration of 0.625 g crude drug / ml was diluted with water to 100 ml in the high-dose and low-dose groups (89.0 ml and 44.5 ml respectively), yielding concentrations of 0.556 g crude drug / ml and 0.278 g crude drug / ml; Lily bulb rehmannia (1:1.43) at a concentration of 1.3225 g crude drug / ml was also diluted with water in the high-dose and low-dose groups. 42 ml and 21 ml of the drug were diluted with water to 100 ml in the high-dose and low-dose groups, respectively, to obtain concentrations of 0.556 g crude drug / ml and 0.278 g crude drug / ml. 1.0 g crude drug / ml of Lilium brownii (1:1) was administered to the high-dose and low-dose groups, respectively, by diluting 55.6 ml and 27.8 ml of the drug to 100 ml, respectively, to obtain concentrations of 0.556 g crude drug / ml and 0.278 g crude drug / ml. Except for the positive control group, all groups were administered the drug by gavage at a volume of 10 ml / kg for 3 consecutive weeks.

[0044] Results: 10 rats were in the sham-operated group and 80 rats were in the PSD model group. They were randomly divided into 8 groups of 10 rats each. The rats were administered the drug as described above for 3 consecutive weeks. Each week or at the end of the administration period, various indicators were tested and samples were collected.

[0045] Example 3 Behavioral experiments.

[0046] Materials: sucrose; cylinder; multifunctional rat spontaneous activity recorder.

[0047] method: ① Sugar Water Preference Test: The purpose was to assess the degree of anhedonia in rats. After the PSD model was established, rats underwent adaptive training. Two bottles of pure water were placed in each cage, and the positions of the water bottles were adjusted daily to eliminate the influence of positional preference. One week before the end of the model, one bottle of 1% sucrose solution and one bottle of pure water were placed in each cage, and the positions of the water bottles were adjusted daily to establish the rats' preference for sugar water, and baseline preference levels were measured. Sugar water preference was tested in each group of rats before drug administration and 1-3 weeks after drug administration. After a 12-hour water deprivation, the rats were fasted, and the test was conducted simultaneously. Two quantitative water bottles were selected, one containing 1% sucrose solution and the other containing pure water. After 12 hours, the water bottles were removed and the rats were weighed, recording the total fluid consumption (sugar water consumption + pure water consumption). The formula for calculating the sugar water preference rate is: Sugar water preference rate (%) = Sugar water consumption / Total fluid consumption × 100%. Total fluid consumption = Sugar water consumption + Pure water consumption.

[0048] ② Forced Swimming Test: The purpose was to assess the behavioral despair state of rats. After the PSD model was established, rats were trained to swim daily. Forced swimming tests were conducted on rats in each group before drug administration and 1-3 weeks after administration. Rats were placed in a stainless steel cylinder 60 cm high, 30 cm in diameter, 45 cm deep, and at a water temperature of 25±2℃. The rats were forced to swim, and their swimming activity was recorded over 6 minutes. When a rat stopped swimming and remained floating motionless with only its nostrils exposed for breathing, or occasionally moved its limbs to prevent sinking, it was recorded as immobile. The cumulative immobile time of the rats floating on the surface was recorded within 5 minutes after collection (the first minute was not recorded as an adaptation and exploration period). After collection, the rats were dried with a towel, warmed to help them regain body temperature, and then returned to their enclosures.

[0049] ③ Spontaneous Activity Test: The purpose is to assess the basic activity ability and exploratory behavior of rats. Spontaneous activity tests were conducted on rats in each group before drug administration and 1-3 weeks after drug administration. The rats were placed in the center of a spontaneous activity box (length, width, and height were 40cm, 40cm, and 50cm, respectively) and the timing was started. The total distance traveled and the movement trajectory of the rats in the spontaneous activity box were recorded in 5 minutes.

[0050] To avoid interference from external factors in the spontaneous activity test, it must be conducted in a quiet environment with a suitable temperature. After each rat's test, its excrement and odor must be thoroughly cleaned before testing the next rat.

[0051] Experimental results on the effects of different ratios of Lilium and Rehmannia glutinosa on improving the behavioral performance of PSD rats are as follows: Figures 2-5 As shown: ① Sugar water preference experiment Figure 2 The graph shows the effect of different ratios of Lilium and Rehmannia glutinosa on the sucrose preference rate of rats in each group. (Based on...) Figure 2It can be seen that, compared with the PSD model group: before administration and 1 week after administration, there was no statistically significant difference in the sucrose preference rate of rats in each dose group with different ratios of Lilium and Rehmannia glutinosa (P>0.05); after 2 weeks of administration, the sucrose preference rate of rats in the (1:1) high dose group of Lilium and Rehmannia glutinosa was significantly increased (P<0.01); after 3 weeks of administration, the forced swimming immobility time of rats in the (1:1) high and low dose groups of Lilium and Rehmannia glutinosa was significantly increased (P<0.01).

[0052] ② Forced swimming experiment Figure 3 The graph shows the effect of different ratios of Lilium brownii and Rehmannia glutinosa on the immobility time of rats in each group during forced swimming. (Based on...) Figure 3 It can be seen that, compared with the PSD model group: before administration, there was no statistically significant difference in the forced swimming immobility time of rats in different dosage groups of Lilium affine with different ratios (P>0.05); after 1 week of administration, the forced swimming immobility time of rats in the (1:1) high-dose group of Lilium affine was significantly reduced (P<0.01); after 2 weeks of administration, the forced swimming immobility time of rats in both the (1:1) high-dose and low-dose groups of Lilium affine was significantly reduced (P<0.01); after 3 weeks of administration, the forced swimming immobility time of rats in the (1:0.5, 1:1.43) high-dose group of Lilium affine and the (1:1) high-dose and low-dose groups of Lilium affine was significantly reduced (P<0.05 and P<0.01).

[0053] ③ Spontaneous activity experiment Figure 4 The graph shows the effect of different ratios of Lilium brownii and Rehmannia glutinosa on the total distance of spontaneous activity in each group of rats. (Based on...) Figure 4 It was found that, compared with the PSD model group: before administration, there was no statistically significant difference in the total spontaneous activity mileage of rats in each dose group of Lilium oxyphylla with different ratios (P>0.05); after 1 week of administration, the total spontaneous activity mileage of rats in the high-dose groups of Lilium oxyphylla (1:0.5, 1:1.43, 1:1) was significantly increased (P<0.01); after 2 weeks of administration, the total spontaneous activity mileage of rats in both the high-dose groups of Lilium oxyphylla (1:0.5, 1:1.43, 1:1) and the low-dose groups of Lilium oxyphylla (1:0.5, 1:1) was significantly increased (P<0.01); after 3 weeks of administration, the total spontaneous activity mileage of rats in each dose group of Lilium oxyphylla (1:0.5, 1:1.43, 1:1) was significantly increased (P<0.01).

[0054] Figure 5 The graph shows the effect of different ratios of Lilium brownii and Rehmannia glutinosa on the spontaneous activity trajectories of rats in each group after 3 weeks of administration. Figure 5 It can be seen that, compared with the PSD model group, after 3 weeks of administration, the spontaneous activity trajectories of rats in each dose group of the Lily and Rehmannia drug pair (1:0.5, 1:1.43, 1:1) were significantly increased and distributed in multiple areas.

[0055] In summary, different ratios of Lilium and Rehmannia glutinosa drugs have significant behavioral improvement effects on rats with post-stroke depression (PSD), manifested as relief of depressive-like behaviors and activity inhibition, with the 1:1 ratio of Lilium and Rehmannia glutinosa drugs being more effective.

[0056] Example 4 The cerebral infarction status, hippocampal neuronal morphology, cell survival, and apoptosis status in rats were detected by TTC staining, Nissl staining, and TUNEL staining of brain tissue.

[0057] Materials: Dissecting microscope; automated tissue dehydrator; paraffin embedding machine; rotary microtome; fully automated intelligent staining machine; constant temperature slide spreader and baker; pathological image processing system; biological microscope; inverted fluorescence microscope; electric thermostatic incubator; sodium pentobarbital; 4% paraformaldehyde; 2% TTC staining solution; Tris-EDTA antigen retrieval solution; Nissl staining solution (toluidine blue method); 95% ethanol; one-step TUNEL apoptosis detection kit (green fluorescence).

[0058] method: ① Brain tissue TTC staining experiment The aim was to assess cerebral infarction in rats. Three weeks after drug administration, the animals were anesthetized and their hearts were perfused with physiological saline and 4% paraformaldehyde. The brains were then harvested and flash-frozen at -20°C for 20 minutes. Slices were cut every 2 mm. The first cut was made at the midpoint of the line connecting the anterior pole of the brain and the optic chiasm; the second cut was made at the optic chiasm; the third cut was made at the infundibulum handle; and the fourth cut was made between the infundibulum handle and the posterior lobe tail pole. The slices were placed in preheated 2% TTC staining solution, wrapped in aluminum foil, and then incubated at 37°C in the dark for 30 minutes, shaking the slices every 10 minutes to ensure thorough staining. Once the slices turned deep red, the staining solution was poured out. The slices were then fixed in formalin and incubated overnight at 4°C in the dark. The slices were then removed for observation and photographs.

[0059] ② Nissl staining The aim was to assess the morphology and number of neurons in the hippocampus of the brain. Paraffin sections were dewaxed to water with xylene and rinsed with distilled water. Sections were then immersed in Nissl stain (toluidine blue method) at 50-60°C for 25-50 minutes. After rinsing with distilled water and 70% ethanol, the sections were differentiated with 95% ethanol until Nissl bodies were clear and the background was adequate. The sections were then rapidly dehydrated with anhydrous ethanol, cleared with xylene, and finally mounted with neutral resin. Images were observed and acquired under an optical microscope for subsequent analysis.

[0060] ③ TUNEL Staining.

[0061] The aim was to assess neuronal apoptosis in the hippocampus of the brain. Paraffin sections were dewaxed and hydrated; washed with PBS, and permeabilized with proteinase K (20 μg / mL) at 37°C for 15 min; washed with PBS, TUNEL assay solution was added, and the sections were incubated in a humidified chamber at 37°C for 60 min in the dark; mounted with DAPI mounting medium, and observed under a fluorescence microscope. Apoptotic cell nuclei showed green fluorescence (FITC), while all cell nuclei showed blue fluorescence (DAPI).

[0062] The effects of Lilium and Rehmannia glutinosa on cerebral infarction, hippocampal neuronal morphology, cell survival, and apoptosis in PSD model rats are as follows: Figures 6-8 As shown: ① Brain tissue TTC staining experiment Figure 6 The image shows the effect (TTC staining) of different ratios of Lilium brownii and Rehmannia glutinosa on cerebral infarction in rats after 3 weeks of administration. Figure 6 It can be seen that, compared with the PSD model group, after 3 weeks of intervention with different ratios of Lilium and Rehmannia glutinosa drug pairs, the cerebral infarction of rats in each dose group was reduced to a certain extent. Among them, the high dose group of Lilium and Rehmannia glutinosa drug pair (1:1) was significantly reduced, and it was basically consistent with the fluoxetine group.

[0063] ②Nissl staining Figure 7 The image shows the effects (Nissl staining) on ​​the morphology of hippocampal neurons in rats after 3 weeks of administration of different ratios of Lilium and Rehmannia glutinosa. Figure 7 It can be seen that, compared with the PSD model group, after 3 weeks of intervention with different ratios of Lilium and Rehmannia glutinosa drug pairs, the number and morphology of neurons in the hippocampus of rats in each dose group were restored to a certain extent, showing abundant Nissl bodies and clear nucleoli. Among them, the high dose group of Lilium and Rehmannia glutinosa drug pair (1:1) showed significant recovery, which was basically consistent with the fluoxetine group.

[0064] ③TUNEL staining Figure 8 The image shows the effects (TUNEL staining) on ​​the survival and apoptosis of hippocampal neurons in rats after 3 weeks of administration of different ratios of Lilium and Rehmannia glutinosa. Figure 8 It can be seen that, compared with the PSD model group, after 3 weeks of intervention with different ratios of Lilium and Rehmannia glutinosa, the apoptosis of nerve cells in the hippocampus of rats in each dose group was inhibited to varying degrees.

[0065] In summary, different ratios of Lilium and Rehmannia glutinosa drug pairs can inhibit neuronal apoptosis to varying degrees, improve the damage status of neurons in the hippocampus, and promote the repair of neuronal structure and function in rats with post-stroke depression (PSD). The 1:1 ratio of Lilium and Rehmannia glutinosa drug pairs is even better.

[0066] Example 5 The levels of inflammatory factors in rat serum, the degree of inflammation in the hippocampus, and the expression and distribution of related proteins were detected by enzyme-linked immunosorbent assay (ELISA), HE staining, Western blotting, and immunohistochemistry.

[0067] Materials: Varioskan LUX multifunctional microplate reader; automated tissue dehydrator; paraffin embedding machine; rotary microtome; fully automated intelligent staining machine; constant temperature slide warmer; pathological image processing system; vertical slab electrophoresis transfer device; trans-blot transfer device; image analysis system; electrophoresis apparatus; Tanon-4200 gel imaging system; rat interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) kits (Quanzhou Ruixin Biotechnology Co., Ltd.); hematoxylin and eosin staining solution (Beijing Regen Biotechnology Co., Ltd.); 4% paraformaldehyde (BioSharp); sodium pentobarbital (Shanghai Institute of Criminal Science and Technology); BCA Protein Assay Kit (Takara); primary antibody: GFAP (mouse-derived) (Affinity). Biosciences); Primary antibody: Iba1 (mouse-derived) (Huaan); Internal control primary antibody: GAPDH (rabbit-derived) (HUABIO); Secondary antibody: HRP-labeled goat anti-mouse IgG, secondary antibody: HRP-labeled goat anti-rabbit IgG (Beiyuntian); Rabbit two-step detection kit, DBA chromogenic kit (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.); GFAP Rabbit mAb, IbalRabbit mAb (Chengdu Zhengneng Biotechnology Co., Ltd.).

[0068] method: ① Serum inflammatory factor detection (ELISA) The aim was to assess the improvement of systemic inflammation in rats. The levels of interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) in rat serum were detected using appropriate enzyme-linked immunosorbent assay (ELISA) kits. Standards and serum samples were added to 96-well plates pre-coated with antibodies and incubated at 37°C. After washing, biotinylated detection antibody, horseradish peroxidase (HRP)-labeled streptavidin, and the chromogenic substrate TMB were added sequentially for further reaction. The reaction was terminated with stop solution. The absorbance (OD) values ​​of each well were measured at 450 nm using a microplate reader. The actual concentrations of inflammatory factors in each sample were calculated based on a standard curve plotted using standard concentrations and OD values.

[0069] ②Hippocampal histopathological examination (HE staining) The aim was to assess the recovery of neuroinflammation in the hippocampus of rat brains. After anesthesia, rats underwent cardiac perfusion and fixation with physiological saline and 4% paraformaldehyde. The fixed tissues were then dehydrated using a routine gradient ethanol treatment, cleared with xylene, and embedded in paraffin. Coronal sections of brain tissue containing the hippocampus were prepared using a paraffin microtome, with a thickness of 5 μm. After dewaxing to water, the sections were stained with hematoxylin and eosin (HE staining) and mounted with neutral resin. The morphology and structure of the hippocampus tissues and the presence of inflammatory cell infiltration were observed under an optical microscope, and images were acquired and analyzed.

[0070] ③Western Blot detection The aim was to assess the expression of relevant proteins. Specific brain regions were harvested and mechanically homogenized using RIPA lysis buffer. After centrifugation at 12,000 rpm for 5 min at 4°C, the supernatant was collected as the total protein extract. Protein concentration was determined using the BCA method: samples were appropriately diluted and reacted with working solution at 37°C for 60 min. Absorbance was measured at 562 nm, and the concentration of each sample was calculated based on the standard curve.

[0071] Equal volumes of protein sample were mixed with 5× loading buffer and boiled for denaturation. SDS-PAGE was performed vertically using a 10% separating gel and a 5% stacking gel (stacking gel voltage 80 V, separating gel voltage 120 V). Subsequently, the protein was transferred to a pre-activated methanol-coated PVDF membrane using a wet transfer method (constant current 200 mA, transfer time 1–3 h).

[0072] After transfer, the gel was blocked with TBST containing 5% skim milk at room temperature for 1 h. It was then incubated overnight at 4°C with the corresponding primary antibody (1:1000 dilution). After washing with TBST, it was incubated with HRP-labeled secondary antibody (1:1000 dilution) at room temperature for 1 h. After thorough washing with TBST, development and image acquisition were performed using an ECL chemiluminescence kit on a gel imaging system. GAPDH was used as an internal control for standardized analysis.

[0073] ④ Immunohistochemical examination The aim was to assess the expression and distribution of relevant proteins. Paraffin sections were dewaxed, hydrated, and antigen-retrieved. Endogenous peroxidase inhibitor was added, and the sections were incubated for 10 min, followed by washing with PBS. Specific primary antibody was added, and the sections were incubated overnight at 4°C. The next day, after washing with PBS, reaction enhancement solution was added, and the sections were incubated at room temperature for 20 min, followed by washing with PBS. Enhancing enzyme-labeled goat anti-rabbit IgG polymer was added, and DAB chromogenic solution was added for staining. After hematoxylin counterstaining of cell nuclei, the sections were routinely dehydrated, cleared, and mounted with neutral resin. Images of different regions of the hippocampus were observed and acquired under an optical microscope.

[0074] Experimental results on the inhibitory effect of Lilium and Rehmannia glutinosa on neuroinflammation in the brain tissue of PSD rats (serum inflammatory factor levels, HE staining of the hippocampus, and detection of GFAP and Iba-1 protein expression and distribution in the hippocampus) are as follows: Figures 9-12 As shown: ①ELISA test Figure 9 The figure shows the effects of different ratios of Lilium and Rehmannia glutinosa on the serum levels of inflammatory factors IL-6, IL-1β, and TNF-α in rats after 3 weeks of administration. Figure 9 It can be seen that, compared with the PSD model group, after 3 weeks of intervention with different ratios of Lilium and Rehmannia glutinosa drug pairs, the serum inflammatory factors (IL-6, IL-1β, TNF-α) levels of rats in each dose group were reduced to some extent (P<0.05 or P<0.01), among which the high dose group of Lilium and Rehmannia glutinosa drug pair (1:1) was significantly reduced (P<0.01), and was basically consistent with the fluoxetine group.

[0075] ② Pathological HE staining examination Figure 10 The image shows the effects (HE staining) of different ratios of Lilium and Rehmannia glutinosa on brain tissue inflammation in rats after 3 weeks of administration. Figure 10 It can be seen that, compared with the same PSD model group, after 3 weeks of intervention with different ratios of Lilium and Rehmannia glutinosa drug pairs, the inflammatory cell infiltration in the hippocampus of the rat brain in each dose group was reduced to a certain extent. The high dose group of Lilium and Rehmannia glutinosa drug pair (1:1) showed the most significant improvement, which was basically consistent with the fluoxetine group.

[0076] ③Western Blot detection Figure 11 The effect of different ratios of Lilium and Rehmannia glutinosa on the expression of GFAP and Iba-1 proteins in the hippocampus of rats in each group after 3 weeks of administration (Western blot results); Figure 12 for Figure 11 The quantitative results are shown in the graph. According to... Figure 11 and Figure 12 It was found that, compared with the PSD model group, after 3 weeks of intervention with different ratios of Lilium and Rehmannia glutinosa drug pairs, the expression level of GFAP protein in the hippocampus of rats in each dose group was significantly increased (P < 0.01); the expression level of Iba1 protein was significantly decreased (P < 0.05 or P < 0.01). Among them, the high-dose group of Lilium and Rehmannia glutinosa drug pair (1:1) was basically consistent with the fluoxetine group.

[0077] ④ Immunohistochemical examination Figure 13 The image shows the effects (immunohistochemical) on the distribution of astrocytes (GFAP) and microglia (Iba1) in the hippocampus of rats in different groups after 3 weeks of administration of different ratios of Lilium brownii and Rehmannia glutinosa. Figure 13 It was found that, compared with the sham-operated group, the expression of glial fibrillary acidic protein (GFAP), a marker of astrocytes, was significantly reduced in the PSD model group rats, while the expression of ionized calcium-binding aptamer 1 (Iba1), a marker of microglia, was significantly increased, suggesting that the PSD model group had pathological changes of reduced astrocytes and increased number / activity of microglia.

[0078] Compared with the PSD model group: After 3 weeks of intervention with different ratios of Lilium brownii and Rehmannia glutinosa, the expression of GFAP (astrocytosis) and the expression of Iba1 (reduced microglia activation) in the hippocampus of rats in all dose groups were upregulated to varying degrees. Among them, the high-dose group of Lilium brownii and Rehmannia glutinosa (1:1) showed the most significant effect, and its expression levels of GFAP and Iba1 were basically consistent with those of the fluoxetine group. In summary, different ratios of Lilium and Rehmannia glutinosa drug pairs have significant anti-neuroinflammatory effects on rats with post-stroke depression (PSD), manifested as systemic inflammation relief and improvement of local hippocampal pathology, with the 1:1 ratio of Lilium and Rehmannia glutinosa drug pairs being more effective.

[0079] Example 6 Western blotting was used to detect signal pathway activation.

[0080] Materials: Vertical slab electrophoresis transfer apparatus; Trans-Blot membrane transfer apparatus; Image analysis system; Electrophoresis apparatus; Tanon-4200 gel imaging system; Primary antibodies: MAPK (rabbit-derived), ERK (rabbit-derived), PI3K (rabbit-derived), Akt (rabbit-derived) (Affinity Biosciences); Internal control primary antibody: GAPDH (rabbit-derived) (Huaan); Secondary antibodies: HRP-labeled goat anti-mouse IgG, HRP-labeled goat anti-rabbit IgG (Beyotime). method: The aim was to assess the expression of relevant proteins. Specific brain regions were harvested and mechanically homogenized using RIPA lysis buffer. After centrifugation at 12,000 rpm for 5 min at 4°C, the supernatant was collected as the total protein extract. Protein concentration was determined using the BCA method: samples were appropriately diluted and reacted with working solution at 37°C for 60 min. Absorbance was measured at 562 nm, and the concentration of each sample was calculated based on the standard curve.

[0081] Equal volumes of protein sample were mixed with 5× loading buffer and boiled for denaturation. SDS-PAGE was performed vertically using a 10% separating gel and a 5% stacking gel (stacking gel voltage 80 V, separating gel voltage 120 V). Subsequently, the protein was transferred to a pre-activated methanol-coated PVDF membrane using a wet transfer method (constant current 200 mA, transfer 1–3 h).

[0082] After transfer, the gel was blocked with TBST containing 5% skim milk at room temperature for 1 h. It was then incubated overnight at 4°C with the corresponding primary antibody (1:1000 dilution). After washing with TBST, it was incubated with HRP-labeled secondary antibody (1:1000 dilution) at room temperature for 1 h. After thorough washing with TBST, development and image acquisition were performed using an ECL chemiluminescence kit on a gel imaging system. GAPDH was used as an internal control for standardized analysis.

[0083] The experimental results of the effects of Lilium and Rehmannia glutinosa on the regulation of the BDNF / TrkB / MAPK / ERK / PI3K / Akt signaling pathway in the hippocampus of PSD model rats are as follows: Figures 14-17 As shown: Figure 14 The figure shows the effect of different ratios of Lilium and Rehmannia glutinosa on the expression of BDNF and TrkB proteins in the hippocampus of rats in each group after 3 weeks of administration (Western blot results). Figure 15 for Figure 14 The quantitative results are shown in the figure. Figure 16 The figure shows the effect of different ratios of Lilium and Rehmannia glutinosa on the expression of MAPK, ERK, PI3K and Akt proteins in the hippocampus of rats in each group after 3 weeks of administration. Figure 17 for Figure 16 The quantitative results are shown in the graph. According to... Figures 14-17 It was found that, compared with the PSD model group, after 3 weeks of intervention with different ratios of Lilium brownii and Rehmannia glutinosa drug pairs, the expression levels of related proteins such as BDNF, TrkB, MAPK, ERK, PI3K, and Akt in the hippocampus of rats in each dose group were significantly increased (P < 0.05 or P < 0.01), except for PI3K protein in the low-dose groups of Lilium brownii and Rehmannia glutinosa drug pairs (1:0.5, 1:1.43). Among them, the high-dose group of Lilium brownii and Rehmannia glutinosa drug pairs (1:1) was basically consistent with the fluoxetine group.

[0084] In summary, different ratios of Lilium and Rehmannia glutinosa drug pairs can all promote the upregulation of the expression of neurotrophic factor BDNF and its receptor TrkB in hippocampal tissue, activate key proteins in downstream MAPK / ERK and PI3K / Akt signaling pathways, and ultimately manifest as behavioral improvement, neuroprotection, inhibition of neuronal apoptosis, reduction of brain inflammation, and activation of neurotrophic pathways. Moreover, the 1:1 ratio of Lilium and Rehmannia glutinosa drug pairs is significantly better than the 1:0.5 and 1:1.43 ratios.

[0085] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The use of a composition of lily and rehmannia in the preparation of a medicament for the prevention and / or treatment of post-stroke depression; said composition being obtained by mixing lily aqueous extract and rehmannia aqueous extract in a volume ratio of 1:0.5, 1:1 or 1:1.

43.

2. The use of a composition of lily and rehmannia in the preparation of a drug for inhibiting neuroinflammation; said composition is obtained by mixing lily aqueous extract and rehmannia aqueous extract in a volume ratio of 1:0.5, 1:1 or 1:1.

43.

3. The application according to claim 2, characterized in that, The inhibition of neuroinflammation includes the inhibition of neuroinflammation by regulating the levels of pro-inflammatory factors TNF-α, IL-1β, and IL-6, and by regulating the expression of GFAP and Iba1 proteins.

4. The use of a composition of lily and rehmannia in the preparation of a drug for inhibiting nerve cell apoptosis; said composition is obtained by mixing lily aqueous extract and rehmannia aqueous extract in a volume ratio of 1:0.5, 1:1 or 1:1.

43.

5. The use of a composition of lily and rehmannia in the preparation of a drug for promoting nerve repair; said composition is obtained by mixing lily aqueous extract and rehmannia aqueous extract in a volume ratio of 1:0.5, 1:1 or 1:1.

43.

6. The use of a composition of lily and rehmannia in the preparation of a drug that increases the expression levels of BDNF, TrkB, ERK, PI3K or Akt proteins; said composition is obtained by mixing lily aqueous extract and rehmannia aqueous extract in a volume ratio of 1:0.5, 1:1 or 1:1.

43.

7. The application according to any one of claims 1 to 6, characterized in that, The preparation method of the lily water extract includes the following steps: mixing lily with water for water extraction to obtain a first residue and a first water extract; mixing the first residue with water for water extraction to obtain a second residue and a second water extract; mixing the first water extract and the second water extract and concentrating to obtain the lily water extract. The preparation method of the Rehmannia glutinosa aqueous extract includes the following steps: mixing Rehmannia glutinosa with water for water extraction to obtain a first residue and a first aqueous extract; mixing the first residue with water for water extraction to obtain a second residue and a second aqueous extract; mixing the first aqueous extract and the second aqueous extract and concentrating to obtain the Rehmannia glutinosa aqueous extract.

8. The application according to claim 7, characterized in that, The concentration of the concentrated lily water extract was 0.5g crude drug / ml.

9. The application according to claim 7, characterized in that, The concentration of the concentrated Rehmannia glutinosa aqueous extract was 1.5g crude drug / ml.

10. The application according to any one of claims 1 to 6, characterized in that, The drug is an oral preparation, which includes tablets, capsules, granules, or oral liquids.

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