Application of osthole in preparation of medicine for protecting cognitive function impairment caused by down-regulation of estrogen receptor

Abstract

The invention relates to the technical field of medicines, in particular to application of osthole in preparation of a medicine for protecting cognitive function impairment caused by down-regulation of an estrogen receptor. The OST has an improvement effect on the learning and memory ability and pathological damage of 6-month-old female peripheral ERs down-regulated mice and ER alpha- / -mouse models. The mechanism of the OST for improving learning and memory may be related to improvement of neuroplasticity, inhibition of nerve apoptosis and enhancement of neurotransmitter by regulating and controlling an estrogen-cholinergic system by the OST. The OST has a better intervention effect on a 6-month-old female ER alpha- / -mouse model, and possibly is a kidney-tonifying medicine which acts on a related target spot of a peripheral target organ so as to participate in regulation of a cognitive function of a central system.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the use of osthol in the preparation of drugs that protect against cognitive impairment caused by estrogen receptor downregulation. Background Technology

[0002] Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by memory loss and cognitive impairment. It is primarily caused by the degeneration of neurons in the hippocampus and cortex, accounting for 60% to 80% of all dementia cases. [1,2] Reports indicate that women account for up to 70% of all AD cases, with an incidence rate 1.5 to 3 times higher than that of men of the same age. This is closely related to the decline in estrogen levels in postmenopausal women. [3,4] Numerous studies have shown that estrogen replacement therapy (ERT) can significantly reduce the incidence of Alzheimer's disease (AD) and improve cognitive function. [5] However, the trend that ERT increases the risk of breast, endometrial, and ovarian diseases significantly limits its clinical application. [6] Currently, the first-line drugs recommended in clinical practice (cholinergic drugs) have limited efficacy due to their single target and lack of regulatory effect on AD in women.

[0003] The primary site of Alzheimer's disease (AD) is in the brain. According to ancient Chinese medicine theory, its main pathological features are "kidney deficiency" and "brain emptiness." The kidneys are the foundation of innate essence and store vital essence; therefore, kidney deficiency leads to essence depletion, and essence depletion results in insufficient production of marrow. [7] The *Suwen* (Plain Questions) chapter "On the Primordial Innocence of Antiquity" states: "At seven years old, a girl's kidney qi is abundant, her teeth change, and her hair grows longer... At five sevens, her yang meridians decline, her face begins to wither, and her hair begins to fall out... At forty-nine, her Ren meridian is deficient, her Chong meridian declines, her Tian Gui (menstrual blood) is exhausted, and her Di Dao (menstrual flow) is blocked, therefore her form deteriorates and she cannot conceive." Thus, a severe decline in kidney essence is one of the important signs in aging women. Furthermore, clinically, the pathogenesis of Alzheimer's disease (AD) in 88% of patients is related to kidney dysfunction. [8] It is evident that older women, a high-risk group for Alzheimer's disease (AD), experience a sharp decline in estrogen levels after menopause, in addition to other factors. [9] Kidney deficiency also plays a crucial role in the development of Alzheimer's disease (AD) in women. Modern Traditional Chinese Medicine research indicates that kidney deficiency is related to an imbalance in the neuroendocrine-immune regulatory network. When brain atrophy occurs in women with AD, the levels of estradiol (E2), pregnenolone, and progesterone are lower than normal. Feria-Romero Iris et al. reported significant changes in estrogen and progesterone gene levels in the brains of ovariectomized AD rats. [9] These studies suggest that the "kidney" in Traditional Chinese Medicine may be closely related to targets in the estrogen regulatory network.

[0004] Estrogen has a wide-ranging effect on the central nervous system, influencing not only reproductive regulation but also cognition, emotion, and neuroprotection. Its effects are mediated by binding to estrogen receptors (ERs)—estrogen receptor α (ERα) and estrogen receptor β (ERβ)—which are widely distributed throughout the brain, including the hippocampus, prefrontal cortex, amygdala, and basal forebrain.

[10] These receptors promote genomic transcriptional regulation and rapid non-genomic signaling, participating in intracellular cascades that influence brain aging and resilience to neurodegenerative stressors.

[11] Estrogen was one of the first hormones recognized for its neuroprotective effects, used to delay or prevent memory changes and neurodegeneration associated with aging, stroke, and Alzheimer's disease.

[12] It has been reported that mice with estrogen deficiency in their brains exhibit spatial memory deficits, and these memory impairments improve when estrogen levels are restored.

[13] .

[0005] Acetylcholine (ACh) is a major neurotransmitter in the brain, active throughout the cortex, basal ganglia, and basal forebrain.

[14] Estrogen and cholinergic signaling pathways are involved in signal transduction related to memory and learning abilities. In Alzheimer's disease (AD) patients, the synthesis, release, and uptake of acetylcholinesterase (ACHE) and choline acetyltransferase (ChAT) are disrupted, often accompanied by neuronal deformation and loss, leading to a decline in learning and memory abilities. Therefore, damage to the cholinergic nervous system is considered one of the causes of AD. Estrogen and cholinergic signaling pathways form an interaction network in the brain, creating a stable balance that jointly regulates cognitive function.

[15] A crucial component of the cholinergic pathway is the basal forebrain cholinergic neurons. The survival and differentiation of these neurons are regulated by nerve growth factor (NGF). NGF binds to its receptor, tyrosine kinase A (TrkA), activating downstream signaling pathways and thus regulating the development and survival of cholinergic neurons. Furthermore, the transcription of the NGF gene is regulated by estrogen. 16] This demonstrates a close interaction between estrogen and the cholinergic pathway, forming a stable balance. However, the sharp drop in estrogen levels after menopause disrupts this balance, potentially a key factor in the development of Alzheimer's disease (AD) in women. Therefore, regulating this balance to prevent and treat AD in women has significant practical implications and broad application prospects.

[0006] Synapses are the biological basis of cognitive function and are involved in hippocampal-dependent learning and memory.

[17] Synaptic damage, including changes in synaptic structure and function, can lead to cognitive impairment.

[18] Synuclein (SYN) is a biomarker for synapse formation, and postsynaptic density protein 95 (PSD95) is a key protein involved in synaptic signal transduction.

[19] Both SYN and PSD95 play crucial roles in synaptic plasticity. Multiple studies have shown that the protein expression of SYN and PSD95 is significantly reduced in the hippocampus of AD mice, while increased expression of these proteins can suppress cognitive decline.

[20] It is also worth noting that brain-derived neurotrophic factor (BDNF), after brain injury, can promote the differentiation and maturation of neurons during the development of the central nervous system and increase synaptic plasticity. 21 This can improve cognitive function.

[0007] Apoptosis is a form of programmed cell death that determines the physiological balance of human organs and systems.

[22] In different individuals with Alzheimer's disease (AD), the expression of the anti-apoptotic factor B-cell lymphoma-2 (bcl-2) was suppressed, while the expression of the apoptotic factors Bcl-2-associated X protein (Bax) and Cleaved-Caspase 3 was upregulated.

[23] This apoptotic neuronal death affects all important areas of the brain, leading to cognitive impairment and memory loss, as well as other symptoms of Alzheimer's disease (AD).

[24] APP protein is cleaved by BACE1 into Aβ protein. The massive aggregation of Aβ will inevitably activate Bcl-2 family pro-apoptotic factors, activating Caspase 3 to form cleaved, active Caspase 3, inducing apoptosis and further aggravating the condition.

[25] Furthermore, the apoptosis regulation mechanism and the synaptic plasticity system together constitute a dual protection system for hippocampal-dependent learning and memory. Their synergistic effect provides a molecular-level bidirectional regulatory basis for the maintenance of cognitive function.

[0008] Osthole (OST), also known as methoxycarpus acetonide, is a coumarin derivative with estrogen-like effects, playing an important role in anti-inflammatory, anticancer, neuroprotective, and immunomodulatory activities.

[26] OST is the mature fruit of *Cnidium monnieri*, a plant in the Apiaceae family. (Li Shizhen) 27] The Compendium of Materia Medica states that "Cnidium monnieri is a medicine for the right kidney, the gate of life, and the qi aspect of the three jiaos," emphasizing its kidney-tonifying and yang-strengthening effects. Modern pharmacological studies have confirmed its definite effect on improving learning and memory functions in the nervous system.

[28] Studies have shown that OST can improve cognitive impairment in APP / PS1 double transgenic AD mice by promoting neural stem cell proliferation and increasing the number of mature neurons. 29] Currently, clinical evidence suggests that OST (osteoscopic stromal prolapse) increases the risk of breast cancer or other cancers. [29-31] Therefore, OST is expected to become an ideal drug for treating AD in women. Summary of the Invention

[0009] To address the aforementioned problems, this invention provides the application of osthol in the preparation of drugs that protect against cognitive impairment caused by estrogen receptor downregulation. This invention is designed to use 6-month-old female peripheral ERs downregulated mice and 6-month-old female ERα... - / - Using mice as the research subjects, this study observed the differences in the effects of OST on cognitive function and brain tissue-related neuroendocrine signaling factors in two models, elucidated the role of the central estrogen and cholinergic pathway balance system in regulating female learning, memory, and cognitive function, and explored whether the kidney-tonifying drug OST participates in the regulation of central cognitive function through peripheral target organ action. This is not only key to improving AD in postmenopausal women, but also beneficial to enriching the scientific connotation of the traditional Chinese medicine theory of "returning to the kidney meridian".

[0010] To achieve the above objectives, the present invention provides the following technical solution:

[0011] This invention provides the application of osthol in the preparation of drugs that protect against cognitive impairment caused by estrogen receptor downregulation.

[0012] Preferably, the osthol improves learning and memory abilities that are downregulated by estrogen receptors.

[0013] Preferably, the osthol improves learning and memory abilities by regulating the estrogen-cholinergic system to enhance neural plasticity, inhibit neuronal apoptosis, and enhance neurotransmitters.

[0014] Preferably, the osthol acts on peripheral target organs to participate in the regulation of central nervous system cognitive function.

[0015] Preferably, the osthol improves the pathological damage caused by downregulation of estrogen receptors.

[0016] Preferably, the osthol alleviates anxiety and depression caused by downregulation of estrogen receptors.

[0017] This invention also provides the application of osthol in the preparation of drugs for treating Alzheimer's disease.

[0018] Preferably, the Alzheimer's disease includes female Alzheimer's disease.

[0019] Preferably, the female Alzheimer's disease includes postmenopausal female Alzheimer's disease.

[0020] The beneficial effects of this invention are:

[0021] 1. OST effect on 6-month-old female peripheral ERs downregulated mice and ERα - / - The study showed improvement in both learning and memory abilities and pathological damage in mouse models.

[0022] 2. The mechanism by which OST improves learning and memory may be related to OST's regulation of the estrogen-cholinergic system, thereby enhancing neural plasticity, inhibiting apoptosis, and strengthening neurotransmitters.

[0023] 3. OST on 6-month-old female ERα - / - The intervention in mouse models showed better results, possibly because the kidney-tonifying drug OST acts on relevant targets in peripheral target organs, thereby participating in the regulation of central cognitive function. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0025] Figure 1 To assess the effect of OST on peripheral ER downregulation in mice during the MWM test, the following data were used: (A) escape latency during the first five days of MWM navigation experiment training; (B) number of platform crossings during the MWM space exploration experiment; (C) time spent in the target quadrant during the MWM space exploration experiment; and (D) representative swimming trajectory of mice on day 6 of the MWM test. All data are expressed as mean ± SEM; n = 10. # P<0.05, ### P<0.001 vs. Control; * P<0.05, ** P<0.01, *** P<0.001 vs. Model.

[0026] Figure 2 To illustrate the effect of OST on the downregulation of peripheral ERs in mice using the NOR and Y-Maze tests, (A) the recognition index in the NOR test, (B) the representative movement trajectory of mice in the NOR test, (C) the spontaneous alternation ratio in the Y-Maze test, and (D) the representative movement trajectory of mice in the Y-Maze test; all data are expressed as mean ± SEM; n = 10. ##P<0.01 vs. Control; * P<0.05, *** P<0.001 vs. Model.

[0027] Figure 3 To assess the effect of OST on the downregulation of anxiety and depression in mice by peripheral ERs, the following data were used: (A) time spent in the open arm during the EPM test; (B) number of times the mouse spent in the open arm during the EPM test; (C) representative movement trajectories of the mouse during the EPM test; (D) immobility time during the FST test. All data are expressed as mean ± SEM; n = 10. ## P<0.01, ### P<0.001 vs. Sham; * P<0.05, ** P<0.01 *** P<0.001 vs. OVX.

[0028] Figure 4 The effect of OST on the pathological morphology of brain tissue in mice with downregulated peripheral ERs (×400). (A) HE staining of brain tissue (CA1, CA3 and cortical area) in mice with downregulated peripheral ERs. (B) Nissl staining of brain tissue (CA1, CA3 and cortical area) in mice with downregulated peripheral ERs. Scale bar represents 20 μm.

[0029] Figure 5 To illustrate the effects of OST on the body weight and uterus of mice with reduced peripheral ERs, (A) body weight of mice in each group with reduced peripheral ERs, (B) representative uterus figures of mice in each group with reduced peripheral ERs, and (C) uterine coefficients of mice in each group with reduced peripheral ERs; all data are expressed as mean ± SEM; n = 8. #### P<0.0001 vs. Control; **** P<0.0001 vs. Model.

[0030] Figure 6 To investigate the effect of OST on the co-expression of ERα, ERβ and ChAT in mice by peripheral ERs downregulation (×400), (A) immunofluorescence was used to detect the expression levels of (CA1)ERα, ERβ and ChAT in the brains of mice with peripheral ERs downregulation.

[0031] Figure 7 To investigate the effect of OST on the downregulation of the estrogen-cholinergic-neurotrophic factor pathway in mice by peripheral ERs, (A) Western blot analysis was performed on the expression levels of ERα(B), ERβ(C), ChAT(D), ACHE(E), NGF(F), and TrkA(G) in mice with downregulated peripheral ERs, n=6. (HI) ELISA kits were used to detect the expression levels of E2(H) and Ach(I), n=4. All data are expressed as mean ± SEM.# P<0.05, ## P<0.01, ### P<0.001 vs. Control; * P<0.05, ** P<0.01, *** P<0.001 vs. Model.

[0032] Figure 8 To investigate the effect of OST on the downregulation of synapse-related proteins and apoptosis proteins in the mouse brain by peripheral ERs, (A) Western blot analysis was performed on the expression levels of PSD95 (B), SYN (C), BDNF (D), Bax (E), and bcl-2 (F) in mice with downregulated peripheral ERs. All data are expressed as mean ± SEM; n = 6. # P<0.05, ## P<0.01 vs. Control; * P<0.05, ** P<0.01, *** P<0.001 vs. Model.

[0033] Figure 9 This is a schematic diagram of the ERα gene sequence.

[0034] Figure 10 For ERα - / - Mouse breeding protocol.

[0035] Figure 11 This is a schematic diagram of the mouse genotype identification results.

[0036] Figure 12 For OST to ERα - / - The effects of the MWM test on mice: (A) escape latency during the first five days of MWM navigation experiment training; (B) number of platform crossings during the MWM space exploration experiment; (C) time spent in the target quadrant during the MWM space exploration experiment; (D) representative swimming trajectory of mice on the sixth day of the MWM test. All data are expressed as mean ± SEM; n = 14. # P<0.05, ## P<0.01, ### P<0.001 vs. Control; * P<0.05, ** P<0.01, *** P<0.001 vs. Model.

[0037] Figure 13 For OST to ERα - / -The effects of NOR and Y-Maze tests on mice: (A) Recognition index in the NOR test; (B) Representative movement trajectory of mice in the NOR test; (C) Spontaneous alternation ratio in the Y-Maze test; (D) Representative movement trajectory of mice in the Y-Maze test; All data are expressed as mean ± SEM; n = 14. ## P<0.01, #### P<0.0001 vs. Control; * P<0.05, ** P<0.01, *** P<0.001 vs. Model.

[0038] Figure 14 For OST to ERα - / - The effects of anxiety and depression on mice: (A) time spent in open arms during the EPM test; (B) number of times mice spent in open arms during the EPM test; (C) representative movement trajectories of mice during the EPM test; (D) immobility time during the FST test; all data are expressed as mean ± SEM; n = 14. # P<0.05, ### P<0.001 vs. Control; * P<0.05, ** P<0.01, *** P<0.001 vs. Model.

[0039] Figure 15 For OST to ERα - / - Effects on mouse brain tissue pathological morphology (×400), ERα - / - HE staining (B) of mouse brain tissue (CA1, CA3 and cortical areas) in various groups - / - Nissl staining of brain tissue (CA1, CA3, and cortical areas) from various mouse groups. Scale bar represents 20 μm.

[0040] Figure 16 For OST to ERα - / - Effects on mouse body weight and uterus, (A)ERα - / - Body weight of mice in each group, (B)ERα - / - Representative uterine images of each mouse group, (C)ERα - / - Uterine coefficients in each mouse group; all data are expressed as mean ± SEM; n = 8. #### P<0.0001 vs. Control; *** P<0.001 vs. Model.

[0041] Figure 17 For OST to ERα - / -Effects of co-expression of ERα, ERβ and CHAT in mice (×400), (A) Immunofluorescence detection of ERα - / - Expression levels of (CA1)ERα, ERβ and ChAT in the brains of mice in different groups.

[0042] Figure 18 For OST to ERα - / - Effects of the mouse estrogen-cholinergic-neurotrophic factor pathway, (A) Western blot analysis of ERα - / - Expression levels of ERα(B), ERβ(C), ChAT(D), ACHE(E), NGF(F), and TrkA(G) in each mouse group, n=6. Expression levels of E2(H) and Ach(I) were detected using a (HI) ELISA kit, n=4. All data are expressed as mean ± SEM. # P<0.05, ## P<0.01, ### P<0.001 vs. Control; * P<0.05, ** P<0.01, *** P<0.001 vs. Model.

[0043] Figure 19 For OST to ERα - / - Effects of synapse-related proteins and apoptosis proteins on expression in mouse brain, (A) Western blot analysis of ERα - / - Expression levels of PSD95(B), SYN(C), BDNF(D), Bax(E), and bcl-2(F) in each mouse group. All data are expressed as mean ± SEM; n = 6. # P<0.05, ## P<0.01, ### P<0.001 vs. Control; * P<0.05, ** P<0.01, *** P<0.001 vs. Model. Detailed Implementation

[0044] The present invention provides the use of osthole in the preparation of a drug for protecting against cognitive function impairment caused by down-regulation of estrogen receptors. In the present invention, the osthole preferably improves the learning and memory ability of estrogen receptor down-regulation. In the present invention, the osthole preferably improves the learning and memory ability by regulating the estrogen-cholinergic system to enhance neuroplasticity, inhibit neuronal apoptosis, and enhance neurotransmitters. In the present invention, the osthole preferably acts on peripheral target organs to participate in the regulation of cognitive function in the central nervous system. In the present invention, the osthole preferably improves the pathological damage of estrogen receptor down-regulation. In the present invention, the osthole preferably alleviates anxiety and depression caused by estrogen receptor down-regulation.

[0045] The present invention also provides the use of osthole in the preparation of a drug for treating Alzheimer's disease. In the present invention, the Alzheimer's disease preferably includes female Alzheimer's disease. In the present invention, the female Alzheimer's disease preferably includes postmenopausal female Alzheimer's disease.

[0046] To further illustrate the present invention, the following examples are used to describe the present invention in detail, but they should not be construed as limiting the protection scope of the present invention.

[0047] Example 1

[0048] Chapter 1 Research on the Learning and Memory and Related Mechanisms of OST in Mice with Down-Regulated Peripheral ERs Section 1 Effects of OST on the Cognitive Ability of Mice with Down-Regulated Peripheral ERs

[0049] 1 Materials and Methods

[0050] 1.1 Experimental Materials

[0051] 1.1.1 Experimental Animals

[0052] In this experiment, 60 6-month-old female C57BL / 6J mice (body weight 20-24 g) were selected and purchased from Jinan Xingkang Experimental Animal Breeding Co., Ltd. (Experimental Animal Use License: SCXK (Lu) 20230005). The mice were housed in a SPF-level laboratory, and the environmental parameters were strictly controlled at a constant temperature (23±1°C) and a constant humidity (50±5%), and a 12h / 12h light-dark cycle lighting system was maintained. The mice were allowed to freely ingest sterilized feed and drinking water, and the sterilized bedding and mouse cages were replaced regularly. The animal experiment was approved by the Animal Experiment Ethics Committee of Shandong First Medical University (NO. 202103030154), and strictly followed the requirements of the Guidelines for the Care and Use of Laboratory Animals.

[0053] 1.1.2 Experimental Instruments

[0054] Table 1 Experimental Instruments and Manufacturers

[0055] Experimental instruments Manufacturer and Model Morris Water Maze Installation TopScan (USA CSI) Y-shaped maze device TopScan (USA CSI) New object recognition device TopScan (USA CSI) Elevated cross-maze device TopScan (USA CSI) Forced swimming device TopScan (USA CSI) Pulsating Vacuum High-Pressure Steam Sterilizer Shandong Xinhua Medical Instrument Co., Ltd., CD-185M

[0056] 1.1.3 Experimental Reagents

[0057] Table 2. Experimental Reagents and Manufacturers

[0058]

[0059]

[0060] 1.2 Experimental Methods

[0061] 1.2.1 Preparation of reagents

[0062] (1) Preparation of sodium pentobarbital solution: Accurately weigh 0.3g of sodium pentobarbital powder and place it in a beaker. Add 100mL of 0.9% sodium chloride solution and stir with a magnetic stirrer until the solution is clear. Transfer it to a 100mL volumetric flask to obtain 0.3% sodium pentobarbital solution. Store in a refrigerator at 4℃ away from light for later use.

[0063] (2) Preparation of OST solution: Refer to Zheng Weina's method for preparing OST solvent.

[32] Furthermore, the ratio of distilled water: Tween-80: anhydrous ethanol was improved to 8:1:1. 375 mg, 750 mg, and 1500 mg of OST powder were accurately weighed and dissolved in 300 mL of solvent respectively. After vortexing, the solutions were placed in an ultrasonic bath for heating and ultrasonication to promote dissolution, thus preparing low-dose (12.5 mg / kg), medium-dose (25 mg / kg), and high-dose (50 mg / kg) OST solutions. 10 mg of E2 powder was accurately weighed and dissolved in 100 mL of solvent to obtain an E2 solution (1 mg / kg).

[0064] (3) Preparation of ICI182780 solution: Accurately weigh 2mg of ICI182780 powder and dissolve it in 1.2% DMSO. Add physiological saline to 20mL. Mix well using a vortex mixer and then place it in an ultrasonic instrument for heating and sonication to promote dissolution. A 1mg / kg ICI182780 solution can be prepared. Prepare it immediately before use.

[0065] 1.2.2 Animal grouping and administration

[0066] Sixty 6-month-old female C57BL / 6J mice were randomly divided into 6 groups: control group, model group (ICI182780), low-dose OST group (OST (12.5 mg / kg) + ICI182780), medium-dose OST group (OST (25 mg / kg) + ICI182780), high-dose OST group (OST (50 mg / kg) + ICI182780), and positive control group E2 group (E2 + ICI182780).

[0067] The model group, low-, medium-, and high-dose OST groups, and the positive control group (E2 group) received intraperitoneal injections of 1 mg / kg of ICI182780 solution for 32 consecutive days. The blank control group received an equal volume of the solvent intraperitoneally. The intraperitoneal injection and gavage administration were performed 30 minutes later. The intraperitoneal injection and gavage administration doses were 0.1 mL / 10 g. The OST group received doses of 12.5 mg / kg, 25 mg / kg, and 50 mg / kg, once daily. The blank control group and model group received an equal volume of the solvent, once daily. The positive control group (E2 group) received 1 mg / kg, administered every two days.

[0068] 1.2.3 Behavioral Experiments

[0069] Mice were administered the drug via gavage for 21 consecutive days, followed by a series of behavioral experiments. The drug was continuously administered throughout the experiments, and the specific procedures were as follows:

[0070] Morris water maze experiment (MWM)

[0071] The Morris water maze test is used to assess the spatial learning and memory abilities of mice.

[33] The mouse MWM (Mixed Motion and Mitigation) experimental setup consisted of a black circular pool (120 cm in diameter and 50 cm in height) and a platform (12 cm in diameter) hidden underwater. Water was poured into the pool to a depth of approximately 30 cm, and a milky white dye was added to color the water, increasing the mice's visibility. The water temperature was maintained at 23 ± 2℃. The pool was divided into four quadrants, with the platform located in the fourth quadrant (1 cm below the water surface). A black blackout cloth was used to cover the pool, with markers of different colors and shapes affixed to the cloth to help the mice memorize the platform's location. The mouse MWM experiment lasted for 6 days and included two parts: a navigational orientation experiment and a spatial exploration experiment.

[0072] ① Orientation and Navigation Experiment: The orientation and navigation experiment lasted for 5 days. Each day, mice were placed into the water sequentially from the center point of each of the four quadrants, facing the pool wall, and allowed to explore freely in the water for 60 seconds. If a mouse found a platform during this period and stayed on it for more than 10 seconds, the time was recorded (escape latency: the time required for the mouse to find the platform). If the mouse did not find the platform within 60 seconds, it was guided to the platform and stayed there for 10 seconds; the escape latency was recorded as 60 seconds. After being removed from the water, the mice were dried with a towel and returned to their cages to warm up.

[0073] ② Spatial Exploration Experiment: Day 6 was the spatial exploration experiment. Before the experiment, the platform was removed, and the mice were placed in the water from the center of the second quadrant, facing the pool wall. They were allowed to explore freely in the water for 60 seconds. The number of times the mice crossed the original platform location and the time spent in the platform's quadrant were recorded. After the mice were removed from the water, they were dried with a towel and returned to their cages to warm up. Data were processed and analyzed using the Topscan software package.

[0074] Novel object recognition (NOR) experiment

[0075] The novel object recognition experiment was used to test non-spatial and short-term memory in mice. The experimental setup consisted of a white box measuring 40cm long, 40cm wide, and 45cm high. The experiment lasted three days and was divided into three phases. Day 1 was the adaptation phase: mice were placed in the box with their heads facing the bottom and allowed free movement for 5 minutes to adapt to the new environment. Day 2 was the exploration phase: two identical red cylinders (old objects) were placed symmetrically on opposite sides of the bottom diagonal of the box, and mice were allowed to explore freely for 5 minutes. Day 3 was the detection phase: one of the red cylinders was replaced with a green cube (new object), and the mouse was placed in the same position and allowed to explore freely for 5 minutes. The tracking data was processed using the Topscan software package, and the novel object recognition index was calculated: Novel object recognition index = (exploration time of new object / (exploration time of new object + exploration time of old object)) × 100%

[34] During the experiment, the surrounding environment was kept quiet. After each experiment, the experimental apparatus was wiped with 75% alcohol to remove odor interference.

[0076] Y-maze experiment

[0077] The Y-shaped maze consists of three arms, each with a 120° angle between them. Each arm measures 35cm × 5cm × 15cm (length × width × height). During the experiment, mice were placed at the central intersection of the three arms and allowed free movement for 10 minutes. The total number of times the mouse entered each arm and the order of entry were recorded. A correct alternation response was defined as the mouse entering all three arms consecutively and without repetition. The number of correct alternations was recorded. The percentage of spontaneous alternations was calculated using the formula: Spontaneous alternation percentage = (Number of correct alternations / (Total number of arm entries - 2)) × 100%

[35] During the experiment, the surrounding environment must be kept quiet, and the experimenter must be out of the mice's sight. After each experiment, the experimental apparatus must be wiped with 75% alcohol to remove odor interference so that the next mouse can be experimented on.

[0078] Elevated plus maze experiment (EPM)

[0079] The elevated cross maze apparatus consists of two open arms and two closed arms. Each arm is 30 cm long and 5 cm wide. Individual open and closed arms are arranged adjacently at a 90° angle, forming a 5 cm long and 5 cm wide square (central area) at their junctions. Mice are placed in the central area with their heads facing the open arms and allowed free movement within the apparatus for 5 minutes. The Topscan software package is used to process the tracking data, recording the number of times and the time the mouse enters the open arms. The calculation formula is as follows: Open arm entry time (%) = Open arm entry time / (Open arm entry time + Closed arm entry time). After each experiment, the apparatus is wiped with 75% alcohol to remove odor interference.

[0080] Forced swimming (FST) test

[0081] The forced swimming experiment apparatus is a transparent hollow cylinder (20cm in diameter and 45cm in height). During the experiment, tap water is added into the cylinder until the water level reaches 15-20cm, at which point the experiment is stopped. The water temperature is maintained at 22±2°C. ℃ C. Place the mice in water and allow them to swim freely for 6 minutes. Analyze the immobile time of the mice from minute 2 to minute 6. After the experiment, wipe the mice clean and return them to their original cages to warm up. Continue to the next set of experiments. Data were processed and analyzed using the Topscan software package.

[0082] 1.2.4 Statistical Analysis

[0083] Experimental results are expressed as mean ± standard error (mean ± SEM), and statistical analysis was performed using GraphPadPrism 8.4.0. The escape latency of the first five days of MWM was analyzed using repeated measures two-way ANOVA to test differences between groups, while other comparisons among multiple groups were analyzed using one-way ANOVA. P < 0.05 was considered statistically significant.

[0084] 2 Results

[0085] 2.1 Effects of OST on learning and memory in mice with downregulated peripheral ERs

[0086] The MWM experiment results showed that during the positioning and navigation experiment, the escape latency of mice in all groups decreased to varying degrees with the increase of training days. On days 4-5 of training, compared with the control group, the escape latency required for the model group to reach the platform was longer (P<0.05). Figure 1 (A) Compared with the Model group, the escape latency of each OST dose group and the positive drug E2 group was shorter, and the differences between the medium and high dose OST groups and the positive drug E2 group and the Model group were statistically significant (P<0.05). Figure 1(A) In the space exploration experiment, compared with the Control group, the Model group had significantly fewer platform crossings and shorter dwell time in the target quadrant (P<0.001). Figure 1 Compared to the Model group, the number of platform crossings and the time spent in the target quadrant were significantly increased in the high-dose OST group and the positive drug E2 group (P<0.01; P<0.05). Figure 1 The trajectory of the space exploration experiment (BC) is as follows: Figure 1 D is shown in the middle.

[0087] The NOR results showed that, compared to the Control group, the new object recognition index of the Model group decreased (P<0.01). Figure 2 In the middle A group, the high-dose OST group and the positive drug E2 group increased the new object recognition index of the Model group (P<0.05). Figure 2 (A). See the representative trajectory diagram. Figure 2 B.

[0088] In the Y-Maze experiment, the spontaneous alternation rate in the Model group was significantly lower than that in the Control group (P<0.05). Figure 2 In the study, different dose groups of OST increased the spontaneous alternation rate in the Model group to varying degrees, with the high-dose OST group showing the most significant improvement (P<0.05). Figure 2 (C). See the representative trajectory diagram. Figure 2 D.

[0089] 2.2 Effects of OST on anxiety and depression in mice with downregulated peripheral ERs

[0090] EPM results showed that, compared with the Control group, the Model group had a reduced open arm dwell time and a reduced number of open arm entries (P<0.01). Figure 3 The high-dose OST group (AB) exhibited anxiety-like behavior; compared with the Model group, the open-arm dwell time and number of open-arm insertions were increased (P<0.01). Figure 3 In the middle (AB), anxiety behavior was relieved; a representative trajectory diagram is shown below. Figure 3 C.

[0091] The FST results showed that, compared with the Control group, the Model group had an increased immobility time (P<0.001). Figure 3 The middle-dose group (D) exhibited depressive-like behavior; compared with the Model group, the immobility time was reduced in the medium- and high-dose OST groups and the positive drug E2 group (P<0.01). Figure 3 The results (D) indicate that OST has a certain alleviating effect on depressive behavior in the Model group.

[0092] 3 Discussion

[0093] This chapter uses the MWM, NOR, and Y-Maze experiments to detect the learning and memory abilities of mice with downregulated peripheral ERs, and uses the EPM and FST experiments to detect anxiety and depression in mice, providing a comprehensive assessment of the behavioral changes in mice.

[0094] The MWM (Wide-Wide Mechanism) is a classic behavioral test for evaluating animal learning ability. MWM results indicated that mice with downregulated peripheral energy expenditure (ERs) exhibited a significant decline in memory. After OST (Optical Characteristic) treatment, the escape latency of these mice decreased, and the number of platform crossings and the time spent in the target quadrant increased on day six. NOR (Novel Orientation) and Y-Maze (YMM) experiments showed a reduction in the novel object recognition index and spontaneous alternation rate in mice with downregulated peripheral ERs, which were significantly increased by OST treatment. These results indicate that the mouse model with downregulated peripheral ERs suffers from learning and memory dysfunction, and that high-dose OST treatment significantly improved the learning and memory abilities of these mice.

[0095] EPM experiments showed that open-arm dwell time and number of open-arm entry times were significantly reduced in mice with downregulated peripheral ERs, but increased after OST treatment. FST experiments showed that immobility time was prolonged in mice with downregulated peripheral ERs, but significantly shortened after OST treatment. In conclusion, mice with downregulated peripheral ERs not only exhibited decreased learning ability but also developed anxiety and depression behaviors. The high-dose OST group had a significant ameliorative effect on anxiety and depression behaviors in mice with downregulated peripheral ERs, providing a theoretical basis for future treatment of AD and comorbid anxiety and depression with OST.

[0096] Section 2. Mechanism of OST's effect on improving learning and memory in mice with peripheral ER downregulation.

[0097] 1. Materials and Methods

[0098] 1.1 Experimental Materials

[0099] 1.1.1 Experimental Apparatus

[0100] Table 3 Experimental Instruments and Manufacturers

[0101] Experimental instruments Manufacturer and Model Organizing the paver Jinhua Technology Co., Ltd., Zhejiang Province, KD-P ice maker Shanghai Anting Scientific Instrument Factory, IMS-130 Fluorescence inverted microscope ZEISS, Axioscope5 Vortex mixer Wuhan Saiweier Biotechnology Co., Ltd., MX-F Horizontal shaking table Shanghai Qite Analytical Instruments Co., Ltd., QT-3 Paraffin slicer Leica Instruments Shanghai Co., Ltd., RM2016 oven Tianjin Laiborui Instrument Co., Ltd., GFL-230 Magnetic stirrer Wuhan Saiweier Biotechnology Co., Ltd. pipette Eppendorf Constant temperature metal bath Hangzhou Borui Technology Co., Ltd., ThermoQ benchtop low temperature centrifuge Eppendorf Electronic balance Sartorius Scientific Instruments, FA3204B Electrophoresis apparatus, electroporation apparatus BIO-RAD (USA), Mini-PROTEAN electromagnetic stirrer Zhencheng Modern Experimental Instruments Co., Ltd., CJJ78-1 Pathology slide machine Leica Instruments Shanghai Co., Ltd., RM2016 Embedding machine Wuhan Junjie Electronics Co., Ltd., JB-P5 Tecan Infinite Microplate Reader TECAN (Switzerland), Infinite200Pro -80℃ ultra-low temperature freezer Haier Corporation (China), DW-86L63 Automated Chemiluminescence Gel Imaging Analyzer GE (USA), Amersham Imager 600

[0102] 1.1.2 Experimental Reagents

[0103] Table 4. Experimental Reagents and Manufacturers

[0104]

[0105]

[0106] Table 5. Experimental Reagents and Manufacturers

[0107] name Manufacturer Item number Mouse AchELISA kit Shanghai Enzyme-Linked Biotechnology Co., Ltd. ml401805 <![CDATA[Mouse E2 ELISA Kit]]> Shanghai Enzyme-Linked Biotechnology Co., Ltd. ml063198 Broad-spectrum rainbow protein maker Thermo Fisher Scientific (China) Co., Ltd. 26616 Horseradish enzyme-labeled goat anti-mouse Beijing Zhongshan Jinqiao Biotechnology Co., Ltd. ZB-2307 Anti-CHAT Sigma-aldrich AB144P DonkeyAnti-Goat IgG H&L Abcam ab150131 Donkey Anti-Mouse IgG H&L Abcam ab150106 Donkey Anti-Rabbit IgG H&L Abcam ab150073 Anti-NGF Abcam ab52918 Anti-ERβ Santa Cruz Biotechnology sc-390243 Blocking with normal donkey serum Wuhan Saiweier Biotechnology Co., Ltd. G1217 DAPI staining reagent (ready-to-use) Wuhan Saiweier Biotechnology Co., Ltd. G1012-10ML 4% paraformaldehyde Wuhan Saiweier Biotechnology Co., Ltd. G1101 HE staining kit Wuhan Saiweier Biotechnology Co., Ltd. G1076 Nissl stain Wuhan Saiweier Biotechnology Co., Ltd. G1036 xylene Sinopharm Group Pharmaceutical Co., Ltd. 10023418 Antigen Repair Buffer Tianjin Guosheng Zhongyuan Technology Co., Ltd. 20230104 Ready-to-use DAPI solution Beijing Solarbio Technology Co., Ltd. 20220829 TSA Fluorescent Double Staining Kit Wuhan Saiweier Biotechnology Co., Ltd. G1235-100T

[0108] 1.2 Experimental Methods

[0109] 1.2.1 Preparation of reagents

[0110] (1) Preparation of lysis buffer: Use a micropipette to draw up high-efficiency RIPA lysis buffer, protein phosphatase inhibitor and PMSF solution, mix the three in a ratio of 100:1:1, mix evenly with a vortex mixer and transfer to a 4°C refrigerator for later use.

[0111] (2) Preparation of 1× electrophoresis buffer: Accurately weigh 144.4g of Glycine powder, 30.3g of Tris-base powder, and 10g of SDS powder using an electronic balance, add pure water to a final volume of 1L, and mix well to obtain 1× electrophoresis buffer. Take 100mL of 10× electrophoresis buffer, add pure water to a final volume of 1L, and mix well to obtain 1× electrophoresis buffer.

[0112] (3) Preparation of 1× electroporation buffer: Accurately weigh 144.4g of Glycine powder and 30.3g of Tris-base powder using an electronic balance, add pure water to a final volume of 1L, mix well, and you will get 10× electroporation buffer. Dilute with 10× electroporation buffer: pure water: methanol = 1:7:2 to get 1× electroporation buffer, and pre-cool at 4℃ for later use.

[0113] (4) Preparation of 1×TBST solution: Use a graduated cylinder to measure 100mL of 10×TBS solution and 800mL of pure water into a beaker, add 2.5mL of Tween-20, and after the magnetic stirrer has mixed thoroughly, transfer the solution to a volumetric flask and add pure water to make up to 1000mL to obtain 1×TBST solution.

[0114] (5) Preparation of 5% blocking solution: Accurately weigh 5.0g of skim milk powder and dissolve it in 100mL of 1×TBST solution. After mixing thoroughly with a magnetic stirrer, a 5% blocking solution is obtained. The blocking solution should be prepared fresh before use.

[0115] (6) Preparation of 10% APS solution: Accurately weigh 1.0 g of APS and dissolve it in 10 mL of pure water. After mixing thoroughly with a magnetic stirrer, a 10% APS solution is obtained. Dispense the solution into 0.5 mL EP tubes and store them in a -20°C refrigerator.

[0116] 1.2.2 Tissue Sampling

[0117] After the behavioral experiments, all mice were anesthetized and blood was collected from their eyes. Three mice were randomly selected from each group. After blood collection, the heart was perfused using 0.9% saline and 4% paraformaldehyde fixative. Following cardiac perfusion, intact brain tissue was carefully removed and fixed in 4% paraformaldehyde for 24 hours to ensure adequate fixation. After fixation, the brain tissue was transferred to paraffin for embedding for pathological staining and immunofluorescence. For the remaining mice, after blood collection from the eyes, the uterus and intact brain were removed. The removed uterus was placed on absorbent paper to remove excess fluid, and then the uterine sample was weighed using an electronic balance. The removed brain tissue, including the hippocampus and cortex, was separated on an ice box, rapidly frozen in liquid nitrogen, and stored at -80°C for subsequent experiments.

[0118] 1.2.3 Paraffin sections

[0119] (1) Dehydration: The brain tissue fixed in paraformaldehyde was removed and rinsed with a low flow of water for 24 hours. Then, graded ethanol was used as a dehydrating agent for soaking to remove excess water from the tissue. The treatment time of the ethanol concentration gradient was as follows: 75% ethanol 60 min, 80% ethanol 45 min, 90% ethanol 45 min, 95% ethanol I 45 min, 95% ethanol II 45 min, 100% ethanol I 45 min, and 100% ethanol II 45 min.

[0120] (2) Transparency: The dehydrated brain tissue was placed in xylene I solution, xylene II solution and xylene III solution in sequence to achieve transparency (30 min each time).

[0121] (3) Paraffin impregnation: The paraffin impregnation operation was carried out in a constant temperature oven at 65°C. The transparent brain tissue sample was immersed in a beaker containing a mixture of paraffin and xylene (ratio = 1:1) for 60 min, and then immersed in paraffin I, paraffin II and paraffin III in sequence (30 min each time).

[0122] (4) Embedding and wax trimming: After the wax is soaked, the brain tissue is slowly placed flat on the mold, liquid paraffin is poured into the mold, and after the paraffin solidifies, the embedding frame is removed. After it has completely cooled, the wax block is trimmed into a trapezoid shape with a blade for later use.

[0123] (5) Sectioning, mounting, and baking: Place the trimmed wax block on the base of the paraffin microtome, ensuring that the cut surface of the wax block is parallel to the blade, the blade is tilted at 15°, and the section thickness is 4μm. Use a brush to pick up the section and flatten it on the microtome, maintaining the microtome water temperature at about 40℃. Select sections without defects and remove them with a glass slide. Then place them in a 60℃ oven overnight to dry and store at room temperature.

[0124] 1.2.4 Hematoxylin-eosin staining (HE)

[0125] (11) Dewaxing and rehydration: The sections were placed in xylene I solution, xylene II solution and xylene III solution in sequence (15 min each time). Then they were transferred to anhydrous ethanol I for 20 min, anhydrous ethanol II for 10 min, 95% ethanol for 10 min, 90% ethanol for 10 min, 80% ethanol for 10 min, 75% ethanol for 10 min, 70% ethanol for 10 min, and rinsed with distilled water for 5 min.

[0126] (2) Staining: Immerse the sections in hematoxylin staining solution for 5 minutes, rinse with running water, continue rinsing after differentiation, immerse the sections in eosin staining solution for 1 minute, and rinse with running water.

[0127] (3) Dehydration and clearing: Soak the slices in 75%, 85%, 100%, 100% and 100% ethanol for 2 min each, and then soak them in xylene I solution and xylene II solution for 5 min each.

[0128] (4) Mounting and observation: Mount the slides with neutral resin and store them at 4°C. Observe the pathological changes in the hippocampus and cortex under a microscope, collect and photograph the slides, and observe the staining.

[0129] 1.2.5 Nissl staining

[0130] (1) Dewaxing and rehydration: Same as Section 1.2.4 HE staining in Chapter 1.

[0131] (2) Staining: Place the slide containing the sample tissue sections into the toluidine blue staining solution for 25 min, while maintaining the temperature of the staining solution at 50-60℃.

[0132] (3) Dehydration and clearing: Immerse the sections in 75%, 80%, 95%, 100%, and 100% ethanol solutions for 1 minute each, and then immerse them in anhydrous ethanol for 2 minutes each. After that, immerse them in xylene I solution and xylene II solution for 5 minutes each.

[0133] (4) Mounting and observation: Mount the slides with neutral resin and store them at 4°C. Observe the volume and number of Nissl bodies under a microscope, collect and photograph them.

[0134] 1.2.6 Immunofluorescence

[0135] (1) Dewaxing and rehydration: Same as Section 1.2.4 HE staining in Chapter 1.

[0136] (2) Antigen retrieval: Tissue slides were placed in EDTA antigen retrieval buffer (pH=9.0) and microwaved for antigen retrieval, first on medium heat for 5 minutes, then on medium-high heat for 5 minutes, and finally on high heat for 5 minutes. During this process, excessive evaporation of the buffer should be prevented, and the slides should not be allowed to dry out. After the slides have cooled naturally, they were placed in PBS (pH=7.4) and washed three times, 5 minutes each time.

[0137] (3) Serum blocking: Place the slide in PBS, shake and wash 3 times, 5 min each time. After washing, gently shake off the excess water on the slide, draw a circle around the tissue with a histochemistry pen to prevent the antibody from flowing away, add goat serum to the histochemistry circle to evenly cover the tissue, and then block at room temperature for 30 min.

[0138] (4) Add the first primary antibody: Gently shake off the blocking solution, drop the diluted primary antibody onto the slide (refer to the respective instructions for the specific dilution ratio of the primary antibody), place the slide flat in a humidified chamber and incubate overnight at 4°C, adding a small amount of water to the humidified chamber to prevent antibody evaporation.

[0139] (5) Add secondary antibody (HRP label): Recover the primary antibody, immerse the slides in PBS and wash and soak them three times on a horizontal shaker for 5 min each time. Dilute the HRP labeling secondary antibody with PBS, add the secondary antibody to the tissue, incubate at room temperature for 50 min, and wash the slides three times with PBS for 5 min each time.

[0140] (6) Add TSA-FITC staining solution: Add TSA-FITC staining working solution to the tissue and incubate at room temperature in the dark for 10 min. Wash with PBS 3 times, 5 min each time.

[0141] (7) Microwave treatment: The tissue sections are placed in a retrieval box filled with antigen retrieval solution and microwave heating is performed to remove the bound primary and secondary antibodies.

[0142] (8) Add the second and third primary antibodies: Dilute the primary antibodies of the other two different species to be detected with PBS, drop them onto the tissue, place the slices flat in a humidified chamber and incubate overnight at 4°C. Add a small amount of water to the humidified chamber to prevent antibody evaporation.

[0143] (9) Add secondary antibody: Place the sections in PBS and wash and soak three times, 3 min each time. After slightly drying the sections, add fluorescent secondary antibody in the circle in the dark, cover the tissue, and incubate at room temperature in the dark for 1 h.

[0144] (10) Counterstaining cell nuclei with DAPI: The sections were placed in PBS and washed and soaked three times, 5 min each time. After the sections were slightly dried, DAPI cell nucleus staining solution was added to the circle and stained at room temperature for 2 min.

[0145] (11) Observation and mounting: The slides were placed in PBS and washed and soaked 3 times, 5 minutes each time. The target antibody and cell nuclear fluorescence were observed under a fluorescence microscope, and the slides were mounted with anti-fluorescence quenching mounting medium.

[0146] 1.2.7 Immunoblotting assay

[0147] protein extraction

[0148] Brain tissue was removed from a -80°C freezer and weighed using a precision electronic balance. The tissue sample and lysis buffer were added to a 1.5 mL centrifuge tube at a ratio of 1 mg tissue to 10 μL lysis buffer. Simultaneously, one 3.0 mm diameter steel ball and two 1.0 mm diameter steel balls were placed in the tube. The sample tube was then fixed in a high-throughput tissue homogenizer, and the homogenization parameters were set to 60 Hz high-frequency oscillation, with a single cycle of 120 s. This homogenization process was repeated twice. Immediately afterwards, the sample tube was transferred to ice (4°C) for lysis for 30 min. After lysis, the centrifuge tube was placed in an ultracentrifuge pre-cooled to 4°C and centrifuged at 13000 rpm / min for 10 min. The supernatant was then stored at -80°C.

[0149] BCA method for determining protein concentration

[0150] (1) Prepare BSA protein standard (0.5 mg / mL): Pipette 20 μL of BSA standard (5 mg / mL) and 180 μL of PBS dilution into an EP tube, mix well and place on ice for later use.

[0151] (2) Preparation of BCA working solution: according to Cu 2+ Prepare the reagent by mixing BCA reagent in a ratio of 50:1. After preparation, mix thoroughly on a vortex mixer (the solution will be light green) and place on ice for later use.

[0152] (3) Set up standard wells: Add the prepared BSA, BCA working solution and PBS to the standard wells in sequence according to Table 6.

[0153] Table 6. Establishment of protein standard curves

[0154] 1 2 3 4 5 6 7 8 Standard concentration (μg / mL) 0 50 100 150 200 300 400 500 PBS (μL) 20 18 16 14 12 8 4 0 BSA (μL) 0 2 4 6 8 12 16 20 BCA working solution (μL) 200 200 200 200 200 200 200 200

[0155] (4) Set up sample wells: Add 1 μL of sample and 19 μL of PBS buffer (sample diluted 20 times) to each well, followed by 200 μL of LCA working solution.

[0156] (5) Incubation: Cover the 96-well plate with the plate lid and incubate in a 37°C water bath for 20-25 minutes.

[0157] (6) Plotting the standard curve: After incubation, the absorbance of the sample at 562 nm was measured using a microplate reader. A regression standard curve was established with the concentration of the protein standard on the x-axis and the absorbance on the y-axis. 2 A value greater than 0.99 is required to calculate the sample protein concentration. The actual sample protein concentration = diluted sample protein concentration × dilution factor.

[0158] protein denaturation

[0159] After calculating the protein concentration of each sample, lysis buffer was added to the samples to make the final protein concentration of each sample equal. 4× loading buffer was added, and the samples were mixed thoroughly. The samples were then placed in a metal bath for high-temperature denaturation at 100℃ for 15 min. After cooling to room temperature, the samples were stored in a refrigerator at -80℃.

[0160] Glue mixing and potting

[0161] (1) Preparation: Align the short glass plate and the long glass plate with a thickness of 1mm, attach them tightly and clip them onto the glue applicator, and ensure that the bottom of the glass plate is tightly connected with the glue strip to prevent glue leakage or liquid surface tilting during the glue pouring process.

[0162] (2) Preparation of separating gel: Based on the molecular weight of the protein, select the appropriate concentration to prepare the separating gel according to Table 7. The values ​​in the table represent the amounts of the two gels used.

[0163] Table 7. Separating Gel Preparation Method

[0164] Reagent (μL) 8% (5mL / block) 10% (5 mL / block) 12% (5 mL / block) Double distilled water 4750 4050 3400 4× Separating Gel Buffer 2500 2500 2500 30% Acrylamide Mixture 2650 3350 4000 10% APS 100 100 100 TEMED 6 4 4

[0165] (3) Preparation of the building block adhesive: Prepare the building block adhesive according to the experimental requirements and Table 8. The values ​​in the table represent the amount of adhesive used for two pieces.

[0166] Table 8. Preparation method of laminating adhesive

[0167] Reagent (μL) Dosage (5mL / tablet) Double distilled water 2294 4×Laminated Gel Buffer 666 30% Acrylamide Mixture 1000 10% APS 40 TEMED 4

[0168] (4) Glue Pouring: After the separating gel is prepared, immediately use a vortex mixer to mix it thoroughly and pour it into the glass tank. When the liquid level reaches 1 cm above the upper edge of the short glass plate, quickly add excess anhydrous ethanol to level the liquid surface. After standing at room temperature for 30 minutes to allow the separating gel to completely solidify, pour out the remaining anhydrous ethanol from the glass tank. Add the prepared building block glue until it slightly overflows the glass tank, insert a dust-free comb to avoid generating air bubbles, and then let it stand at room temperature for 25 minutes to allow the building block glue to completely solidify before use.

[0169] Sample loading and electrophoresis

[0170] (1) Installation: Install the prepared gel plates onto the gel plate holder, with the short glass plate facing inward and the long glass plate facing outward. Place the gel plate holder into the electrophoresis tank filled with 1× electrophoresis solution.

[0171] (2) Sample loading: Slowly pull out the comb horizontally, take out the sample stored in the -80℃ refrigerator, mix it with a vortex shaker, and add the sample and marker into the corresponding sample loading well.

[0172] (3) Electrophoresis: Turn on the power and set the electrophoresis conditions to a constant voltage of 80V to start electrophoresis. After the marker bands have completely separated, adjust the voltage to a constant voltage of 160V to accelerate the electrophoresis. Observe the electrophoresis of bromophenol blue. Stop the electrophoresis when the bromophenol blue is close to the bottom of the glass plate.

[0173] Transfer membrane (wet transfer)

[0174] Activate the polyvinylidene fluoride (PVDF) membrane in methanol for 30-60 seconds, then immediately immerse it in pre-cooled 1× transfer buffer at 4°C. After electrophoresis, remove the gel, trim off excess gel, and gently transfer the desired gel onto a transfer clamp lined with filter paper. Cover with the PVDF membrane and gently close the transfer clamp. Insert the transfer clamp into the transfer tank, aligning the positive and negative electrodes. Add 1× transfer buffer to the designated mark in the transfer tank, then place it in a basin of ice water to prevent overheating during transfer. Set the transfer conditions to a constant current of 240 mA for 60-90 minutes, based on the molecular weight of the target protein.

[0175] Blocking and antibody incubation

[0176] Prepare an antibody incubation chamber and pour in 5 mL of 5% blocking buffer. Place the transferred PVDF membrane face up in the incubation chamber. Block on a horizontal shaker at 56 rpm for 2-4 hours at room temperature. After blocking, discard the blocking buffer and immediately add the corresponding primary antibody. Incubate overnight on a horizontal shaker at 4°C. After recovering the primary antibody, wash the PVDF membrane with 1×TBST at 100 rpm for 5 minutes, 4 times. Select the appropriate secondary antibody based on the species from which the primary antibody is derived. After washing, add the secondary antibody and incubate on a horizontal shaker at 56 rpm for 60 minutes at room temperature. Recover the secondary antibody and repeat the washing procedure 4 times.

[0177] ECL Immunoassay Immunoassay

[0178] Prepare a developer solution (A:B = 1:1) and drop a small amount evenly onto the PVDF membrane. Expose and scan the membrane in a developer apparatus for imaging and development. Analyze the grayscale values ​​using ImageJ software and calculate the expression level of the sample protein using the following formula: Sample protein expression level = Grayscale value of target protein / Grayscale value of internal reference protein

[0179] 1.2.8 Enzyme-linked immunosorbent assay

[0180] (1) Sample preparation: The tissue was added to PBS solution at a ratio of 1:9 and then ground thoroughly with a low-temperature grinder. The grinding was repeated twice. The sample was placed on ice and lysed for 30 min. Then, it was centrifuged at 5000 rpm for 10 min at 4℃. The supernatant was used for subsequent detection.

[0181] (2) Preparation: Take the kit out of the 4℃ refrigerator in advance and let it equilibrate at room temperature for 60 minutes. Take the 96-well plate pre-coated with capture antibody out of the aluminum foil bag for later use.

[0182] (3) Sample addition: Pre-divide the wells into standard wells, blank control wells, and sample wells. Add 50 μL of standard at different concentrations (arranged from low to high concentration) to the standard wells and set up duplicate wells. Add 50 μL of the sample to be tested to the sample wells and set up duplicate wells in the same way. Do not add standard or sample to the blank control wells.

[0183] (4) Adding enzyme: Add 100 μL of horseradish peroxidase (HRP) labeled detection antibody to the standard wells and sample wells respectively. Then cover the well plate with a sealing film and place it in a 37°C water bath for 60 min in the dark.

[0184] (5) Washing: After removing the well plate from the water bath, carefully peel off the sealing film, discard the liquid, spin dry, add 350uL of washing solution to each well, let stand for 60s and then discard, repeat this 5 times, and pat dry.

[0185] (6) Color development: After washing, add 50 μL of substrate A solution to each well, then add 50 μL of substrate B solution, attach the sealing film, wrap with aluminum foil, and react in a 37°C water bath in the dark for 15 min.

[0186] (7) Termination: After removing the plate from the water bath, add 50 μL of termination solution to each well to terminate the reaction. At this time, the solution changes from blue to yellow.

[0187] (8) Detection: Within 15 minutes after adding the stop solution, use an ELISA reader to read the absorbance (OD) value at a wavelength of 450 nm.

[0188] (9) Statistics: In the Excel worksheet, use the standard concentration as the horizontal axis and the corresponding OD value as the vertical axis, and use the blank control well as the 0 well. Draw the linear regression curve of the standard according to the instructions, and calculate the concentration value of each sample according to the curve equation.

[0189] 1.2.9 Statistical Analysis

[0190] Experimental results are expressed as mean ± standard error (mean ± SEM), and statistical analysis was performed using GraphPad Prism 8.4.0. One-way ANOVA was used to test differences between groups. P < 0.05 was considered statistically significant.

[0191] 2 Results

[0192] 2.1 Effects of OST on the pathological morphology of brain tissue in mice with downregulated peripheral ERs

[0193] HE staining results showed that in the Control group, the cellular structure of the CA1, CA3, and cortical areas was normal, with neurons tightly packed and morphologically intact; in the Model group, the neuronal cytoplasm was deeply stained, disordered, and loosely structured; after OST treatment, the cell architecture was restored, and the cells were more tightly packed. Figure 4 The results (A) indicate that OST can, to some extent, improve neuronal loss and tissue structure disorder induced by peripheral ER downregulation in mice.

[0194] Nissl staining results showed that in the Control group, Nissl bodies were abundant, tightly arranged, and morphologically healthy in the CA1, CA3, and cortical regions; in the Model group, Nissl bodies were loosely arranged, and the cytoplasm was darker, indicating neuronal damage and degeneration; the number and morphology of Nissl bodies were improved in all OST dose groups. Figure 4 (B) suggests that OST may have a positive effect on the functional recovery of neurons.

[0195] 2.2 Effects of OST on body weight and uterus in mice with downregulated peripheral ERs

[0196] During the experiment, we monitored the body weight of the mice in each group. The results showed that there was no significant change in body weight among the groups over time. Figure 5 (A). Simultaneously, we observed changes in the morphology and weight of the mouse uterus. The results showed that, compared to the Control group, the uterus in the Model group was significantly atrophied, with varying degrees of recovery after OST and drug administration, tending towards a normal morphology. Figure 5 (B) Compared to the Control group, the uterine weight in the Model group was significantly reduced (P<0.0001). Figure 5 After administration of C and E2, uterine weight increased significantly (P<0.0001). Figure 5 (C)

[0197] 2.3 Effects of OST on the estrogen-cholinergic-neurotrophic factor pathway in the hippocampus of mice with peripheral ERs downregulating

[0198] Immunofluorescence results showed that, compared with the control group, the expression of ERα, ERβ, and ChAT in the CA1 region of the hippocampus was significantly decreased in the model group. Figure 6 (A); Compared with the Model group, the expression of ERα, ERβ and ChAT in the high-dose OST group and the positive drug E2 group was significantly increased. Figure 6 (A)

[0199] Western blot results showed that, compared with the control group, the expression of ERα, ERβ, ChAT, NGF, and TrkA was decreased in the model group (P<0.05). Figure 7 In the model group (AD, FG), the expression of ACHE was increased (P<0.01). Figure 7 In the study of protein A and E, expression of all the aforementioned proteins was reversed after OST treatment, with statistically significant differences between the high-dose OST group and the Model group (P<0.05). Figure 7 (AG). ELISA kit results showed that, compared with the Control group, the expression of E2 and Ach in the Model group was downregulated (P<0.05). Figure 7 In the high-dose OST group (HI), compared with the Model group, the expression of E2 and Ach was significantly upregulated (P<0.05). Figure 7 (China HI).

[0200] 2.4 Effects of OST on the expression of hippocampal synapse-related proteins and apoptosis proteins downregulated by peripheral ERs in mice

[0201] We used Western blot to detect the expression of synapse-related proteins PSD95, SYN, and BDNF, as well as apoptosis-related proteins Bax and bcl-2 in the hippocampus of mouse brain tissue. The results showed that, compared with the control group, the expression of PSD95, SYN, and BDNF was significantly reduced in the model group (P<0.05). Figure 8 Compared to the Model group, the expression of the above proteins was significantly increased in the high-dose OST group (P<0.01). Figure 8 (AD). Compared with the control group, the expression of Bax was upregulated and the expression of bcl-2 was downregulated in the model group (P<0.05). Figure 8 In the high-dose OST group (A, EF), compared with the Model group, the expression of Bax and bcl-2 was reversed (P<0.01). Figure 8 Middle A, EF).

[0202] 3 Discussion

[0203] ERα is the core and dominant mediator of estrogen regulation of uterine development, ensuring the normal growth and structural establishment of the uterine myometrium and endometrium. Its deficiency can lead to severe uterine hypoplasia or even atrophy. ERβ, on the other hand, plays a synergistic regulatory role. Both are crucial for maintaining normal uterine development.

[36] The results of this study showed that the mouse model group with downregulated peripheral ERs exhibited uterine atrophy and a reduced uterine coefficient, demonstrating that peripheral ERs were suppressed after injection of ICI182780. These characteristics were improved to some extent after OST treatment, revealing the estrogen-like effect of OST.

[0204] Memory loss in AD is thought to be associated with central cholinergic dysfunction in the basal forebrain region, the origin of cholinergic neural circuits that project to multiple brain regions, including the cerebral cortex and hippocampus.

[37] The typical manifestations are elevated ACHE activity and decreased ChAT expression.

[38] Mounting evidence suggests that altered TrkA / p75 expression, due to NGF deficiency, leads to cholinergic metabolic dysfunction in the basal forebrain and promotes Aβ deposition. [39,40] This study further found that peripheral ER downregulation led to decreased levels of E2, ERα, and ERβ in the mouse brain, accompanied by an imbalance in the expression of Ach, ChAT, ACHE, NGF, and TrkA. Similarly, immunofluorescence results showed a significant decrease in the co-expression of ERα, ERβ, and ChAT in the hippocampus of the Model group. These experimental results suggest that estrogen deficiency may impair cholinergic neuron function. OST intervention in mice restored E2 levels and the ERα / ERβ-ChAT signaling network in the Model mouse brain, correcting Ach metabolic imbalance and NGF-TrkA pathway abnormalities. This suggests that OST, through its estrogen-like effects, forms a network with cholinergic signaling pathways in the brain, jointly regulating cognitive function.

[0205] Deficiencies in presynaptic SYN and postsynaptic PSD95 are associated with cognitive decline in AD.

[41] Changes in synaptic function and the degree of synaptic loss in AD patients are closely related to the severity of cognitive impairment. Decreased expression of BDNF, PSD95, and SYN is the pathological basis of AD.

[42] The results of this study showed that peripheral ERs significantly reduced the expression of BDNF, PSD95, and SYN in the mouse brain. OST treatment increased the expression levels of these proteins, with higher doses showing better results. In vitro studies also showed that OST treatment could prevent the reduction of PSD95 and SYN in APP-induced BE(2)-M17 cells, possibly through upregulation of miR-132 and inhibition of APP expression.

[43] .

[0206] Notably, our study found that peripheral ER downregulation in the hippocampus of mice exhibited apoptosis imbalance: increased expression of the pro-apoptotic protein Bax and decreased activity of the anti-apoptotic protein bcl-2. OST intervention restored the expression of both Bax and bcl-2. This indicates that OST can inhibit the expression of cleaved Caspase-3, increase the bcl-2 / Bax ratio, inhibit neuronal apoptosis, and improve cognitive function in APP / PS1 mice.

[44] In summary, OST may improve learning and memory abilities by upregulating estrogen levels, enhancing synaptic plasticity, inhibiting neuronal apoptosis, and repairing the cholinergic system.

[0207] summary

[0208] Mice with downregulated peripheral ERs exhibited learning and memory dysfunction and pathological damage, which improved after OST treatment. The mechanism of action of OST may be through regulating the estrogen-cholinergic pathway, thereby enhancing synaptic plasticity, inhibiting apoptosis, and enhancing neurotransmitter expression, thus improving learning and memory abilities.

[0209] Example 2

[0210] Chapter Two OST for ERα - / - Effects on mouse learning and memory and related mechanisms

[0211] Section 1 OST vs ERα - / - Effects on cognitive abilities in mice

[0212] 1ERα - / - Mouse establishment

[0213] 1.1 Construction of ERα gene knockout mice using CRISPR-Cas9 technology

[0214] The combination of CRISPR and Cas9, along with small guide RNA (sgRNA), guides the Cas9 nuclease to a designated genomic site. Cas9 then cleaves the target gene, breaking the DNA double strand, thus achieving site-specific editing of genomic DNA. The combination of CRISPR and related Cas9, via pronuclear microinjection, provides an effective and simple method for creating genetically engineered mice. 45] .

[0215] 1.1.1. Preparation of ERα gene knockout mice

[0216] The sequence of the ERα gene (NM_001302531.2) of C57BL / 6 mice was retrieved from NCBI (https: / / www.ncbi.nlm.nih.gov). Since the first exon is missing an amino acid, exons were counted starting from the second exon. sgRNA was designed using GenScript's sgRNA construction system, and the target sites are shown in Table 9.

[0217] Table 9. Details of the designed sgRNA sequence

[0218]

[0219] After comprehensive consideration, sgRNA2 and sgRNA8 were chosen, and their positions in the transcript are as follows: Figure 9 As shown:

[0220] Target cell delivery: Cas9 / sgRNA was microinjected into fertilized eggs of ICR mice obtained through in vitro fertilization, and its genes were edited. The cells were then transplanted into surrogate female mice to breed transgenic mice. Stable, genetically inherited parental ERα was used. + / - Mice were subsequently bred to obtain the ERα required for the experiment. + / + and ERα - / - Mice.

[0221] 1.1.2ERα - / - Subsequent feeding and breeding program for mice

[0222] like Figure 10 The ERα shown - / - Mouse reproduction.

[0223] 1.1.3 Genotyping of ERα gene knockout mice

[0224] Three weeks after birth, mice were numbered and labeled, and their tails were cut off, with a length of approximately 0.3-0.5 cm. The tails were stored in EP tubes and sent to Qingke Biotechnology Co., Ltd., where Sanger sequencing technology was used to identify the mouse genotype. Based on the gene testing results, ERα mice were screened. + / + and ERα - / - Mice, Figure 11 The diagram shows the sequencing peaks for genotype identification. From top to bottom, they represent homozygous deletion, heterozygous deletion, and no deletion.

[0225] 2 Materials and Methods

[0226] 2.1 Experimental Materials

[0227] 2.1.2 Laboratory Animals

[0228] This experiment used 70 six-month-old female ERα mice. - / -Mice were used in the experiments, with wild-type (WT) mice of the same age and genetic background serving as controls. Mice were bred and housed in an SPF-grade laboratory at a temperature of 23±2°C. ℃ Temperature: 50±10% humidity, 12-hour light / dark cycle. Mice were allowed free access to food and water, and sterilized bedding and cages were changed regularly. Animal experiments were approved by the Animal Experiment Ethics Committee of Shandong First Medical University (NO.202103030154) and strictly followed the guidelines for the care and use of laboratory animals.

[0229] 2.1.3 Experimental Apparatus

[0230] The experimental equipment is the same as in Chapter 1, Section 1.

[0231] 2.1.4 Experimental Reagents

[0232] The experimental reagents are the same as those in Chapter 1, Section 1.

[0233] 2.2 Experimental Methods

[0234] 2.2.1 Preparation of reagents

[0235] The preparation method for the reagents is the same as in Chapter 1, Section 1.

[0236] 2.2.2 Animal grouping and administration

[0237] 70 6-month-old female ERα - / - Mice were randomly divided into 5 groups: Model, low-dose OST (12.5 mg / kg), medium-dose OST (25 mg / kg), high-dose OST (50 mg / kg), and positive control group E2 (1 mg / kg). WT mice with the same genetic background served as the blank control group.

[0238] The drug was administered via gavage for 32 consecutive days at a dose of 0.1 mL / 10 g. The doses for the OST groups were 12.5 mg / kg, 25 mg / kg, and 50 mg / kg, once daily. The blank control group and the model group were given an equal volume of solvent once daily. The dose of the positive control drug E2 was 1 mg / kg, administered every two days.

[0239] 2.2.3 Behavioral Experiments

[0240] Mice were administered the drug via gavage for 21 consecutive days, followed by a series of behavioral experiments. The drug was continuously administered throughout the experiments, and the specific procedures were as follows:

[0241] MWM

[0242] Because the experimental animals are of different species, fill the pool with clean water to a depth of about 30cm. The rest of the operation is the same as in the first section of Chapter 1.

[0243] NOR

[0244] The experimental procedure is the same as in Chapter 1, Section 1.

[0245] Y-Maze

[0246] The experimental procedure is the same as in Chapter 1, Section 1.

[0247] EPM

[0248] The experimental procedure is the same as in Chapter 1, Section 1.

[0249] FST

[0250] The experimental procedure is the same as in Chapter 1, Section 1.

[0251] 2.2.4 Statistical Analysis

[0252] The statistical analysis methods are the same as those in Chapter 1, Section 1.

[0253] 3 Results

[0254] 3.1OST to ERα - / - Effects of mouse learning and memory

[0255] MWM results showed that during the positioning and navigation experiment on days 1-5, the escape latency of mice in all groups decreased with increasing training days. On days 3-5, the escape latency of the model group was significantly higher than that of the control group, while the escape latency of the medium- and high-dose OST groups and the positive control drug E2 group was significantly lower than that of the model group (P<0.05). Figure 12 (A) In the space exploration experiment on day 6, the model group had significantly fewer platform crossings and shorter time spent in the target quadrant compared to the control group (P<0.01). Figure 12 In the OST medium- and high-dose groups and the positive drug E2 group, the number of platform crossings and the time spent in the target quadrant were significantly higher than those in the model group (P<0.05). Figure 12 (See the representative trajectory diagram of the space exploration experiment in the middle BC). Figure 12 D.

[0256] The NOR results showed that the new object recognition index of the model group was significantly lower than that of the control group (P<0.01). Figure 13 In the study of OST (A), the new object recognition index of different dosage groups and the positive drug E2 group was significantly higher than that of the model group (P<0.05). Figure 13 (A). Representative trajectory diagrams are shown in [reference 1]. Figure 13 B.

[0257] Similarly, Y-Maze results showed that the spontaneous alternation ratio in the model group was significantly lower than that in the control group (P<0.0001). Figure 13In the study of OST (C), the spontaneous alternation ratio in different dose groups was significantly higher than that in the model group (P<0.05). Figure 13 (C). Representative trajectory diagrams are shown in [reference needed]. Figure 13 D.

[0258] 3.2OST to ERα - / - Effects of anxiety and depression on mice

[0259] EPM results showed that, compared with the control group, the open arm dwell time and the number of open arm insertions were significantly reduced in the model group (P<0.001). Figure 14 Compared with the model group, the number of intraoperative insertions and the duration of intraoperative insertion increased in the medium- and high-dose OST groups and the positive drug E2 group (P<0.05). Figure 14 (AB). Representative trajectory diagrams are shown below. Figure 14 The above results indicate that OST may improve ERα. - / - Anxiety-like behavior in mice.

[0260] FST results showed that, compared with the control group, the immobility time in the model group was significantly increased (P<0.001). Figure 14 Compared with the model group, the immobility time in the medium and high dose OST groups was reduced and showed a dose-dependent effect (P<0.01). Figure 14 (D). The above results indicate that OST may improve ERα. - / - Depressive-like behavior in mice.

[0261] 4 Discussion

[0262] In the rodent brain, ERα is mainly distributed in the hippocampus, preoptic lobe, and hypothalamus, areas involved in autonomic function, emotion regulation, and association and emotional memory. (Wang Jishen et al.) 46] Discovery of ERα - / - The prolonged escape latency in the water maze in mice and the increased production of Aβ in the cerebral cortex and hippocampus confirmed that the absence of ERα can promote the pathological progression of AD.

[0263] This chapter mainly observes the behavioral performance of mice through MWM, NOR, and Y-Maze experiments, as well as EPM and FST experiments. The results of this study show that 6-month-old female ERα - / - Mice exhibited significant learning and memory impairment in the MWM, NOR, and Y-Maze tests, with medium and high doses of OST showing the best improvement in these tests. Further research revealed that 6-month-old female ERα... - / - The mice also exhibited anxiety-depression-like behaviors, and medium and high doses of OST had a significant restorative effect on these behaviors.

[0264] Section 2 OST vs ERα - / - Mechanism study of the effect of improving learning and memory in mice

[0265] 1. Materials and Methods

[0266] 1.1 Experimental Materials

[0267] 1.1.1 Experimental Apparatus

[0268] The experimental equipment is the same as in Chapter 1, Section 2.

[0269] 1.1.2 Experimental Reagents

[0270] The experimental reagents are the same as those in Chapter 1, Section 2.

[0271] 1.2 Experimental Methods

[0272] The procedures for HE staining, Nissl staining, immunofluorescence, Western blotting, enzyme-linked immunosorbent assay (ELISA), and statistical analysis are the same as those in Section 2 of Chapter 1.

[0273] 2 Results

[0274] 2.1OST to ERα - / - Effects of histopathological morphology on mouse brain tissue

[0275] HE staining results showed that in the Control group, cells in the CA1 region were regularly arranged, with uniformly stained nuclei and prominent nucleoli; neurons in the CA3 region had high density and plump cell bodies; the boundary between the nucleus and cytoplasm in the cortex region was clear, with no signs of inflammatory infiltration. In the Model group, cells in the CA1 region were smaller, with pyknosis and deep staining of the nuclei; neuronal density in the CA3 region was decreased, with localized vacuolar degeneration; interstitial edema and eosinophilic bodies were observed in the cortex region. The pathological damage in the OST-treated group and the positive control group (E2) was significantly alleviated, with clearer cell outlines, more regular arrangement, and a trend towards the normal group. See details. Figure 15 A.

[0276] Nissl staining results showed that in the Control group, Nissl bodies were evenly distributed, plump, and had clear nuclei in the hippocampal CA1, CA3, and cortical regions, without abnormal vacuolation or atrophy. In the Model group, the number of Nissl bodies in some brain regions was reduced, their arrangement was disordered, and cell bodies were atrophied. The Nissl body density significantly increased in the OST and E2 treatment groups, Nissl body granules re-aggregated, and nucleolar clarity improved. See details... Figure 15 B.

[0277] 2.2OST to ERα - / - Effects on mouse body weight and uterus

[0278] During the experiment, we monitored the body weight of mice in each group. The results showed that, over time, the Model group gained weight relative to the Control group ( Figure 16 (A). Simultaneously, we observed changes in the morphology and weight of the mouse uterus. The results showed that, compared to the Control group, the uterus in the Model group was significantly atrophied, and after administration of OST and the positive control drug E2, it recovered to varying degrees, tending towards a normal morphology. Figure 16 (B) Compared to the Control group, the uterine weight in the Model group was significantly reduced (P<0.0001). Figure 16 After administration of C and E2, uterine weight increased significantly (P<0.001). Figure 16 (C)

[0279] 2.3OST to ERα - / - Effects of the estrogen-cholinergic-neurotrophic factor pathway in the mouse hippocampus

[0280] Immunofluorescence results showed that, compared with the control group, the model group showed almost no ERα expression in the hippocampal CA1 region, and ERβ expression was downregulated. After treatment with a high dose of OST, ERβ expression was reversed. Figure 17 (A) Compared with the control group, the expression of ChAT in the CA1 region of the hippocampus was significantly downregulated in the model group, and the expression of ChAT was reversed after treatment with medium and high doses of OST. Figure 17 (A)

[0281] Western blot results showed that, compared with the control group, the expression of ERα in the hippocampus of the model group was significantly reduced, proving that ERα expression was significantly reduced. - / - The mouse model was successfully established. Furthermore, the expression of ERβ in the hippocampus of the model group showed an upregulation trend, which further increased after OST treatment. Figure 18 The expression levels of ChAT, NGF, and TrkA were all downregulated to varying degrees, while the expression of ACHE was upregulated (P<0.01). Figure 18 In patients with high-dose OST (digestive disease), the expression levels of ChAT, NGF, and TrkA were downregulated, and the expression of ACHE was downregulated (P<0.05). Figure 18 DG (China)

[0282] Meanwhile, the ELISA kit results showed that, compared with the control group, the expression of E2 in the hippocampus of mice in the model group showed an upregulation trend, but this was not statistically significant (P>0.05). Figure 18 (H). Furthermore, compared to the control group, the expression of Ach in the hippocampus of the Model group mice was significantly downregulated (P<0.01, H). Figure 18(I); After low and high doses of OST treatment, the expression level of Ach was significantly upregulated (P<0.01, Figure 18 Middle I).

[0283] 2.4OST to ERα - / - Effects of mouse hippocampal synapse-related protein and apoptosis protein expression

[0284] Western blot results showed that, compared with the control group, the expression of hippocampal synapse-related proteins PSD95, SYN, and BDNF was reduced in the model group (P<0.01). Figure 19 High doses of OST significantly increased the expression of the above proteins (P<0.05). Figure 19 (AD). Compared with the control group, the Model group showed increased Bax expression and decreased bcl-2 expression (P<0.01). Figure 19 High doses of OST effectively reversed the expression of Bax and bcl-2 (P<0.05). Figure 19 Middle A, EF).

[0285] 3 Discussion

[0286] 6-month-old female ERα - / - The mice exhibited significant learning and memory impairments, as well as anxiety and depression. Further pathological examination revealed typical Alzheimer's disease (AD) neuropathological features in the hippocampus and cortex, including neuronal swelling, nuclear pyknosis, and cell loss. Notably, OST intervention effectively alleviated these pathological damages, suggesting its potential therapeutic potential in improving AD neurodegenerative changes.

[0287] Cholinergic system imbalance and synaptic plasticity impairment are core components of the AD pathological process. This study found that 6-month-old female ERα - / - In mice, Ach metabolism was disordered (decreased Ach levels, dysregulated ChAT and ACHE expression), and NGF-TrkA signaling was significantly inhibited. Simultaneously, significant downregulation of hippocampal synapse-related proteins PSD95, SYN, and BDNF reflected impaired synaptic structural and functional plasticity, consistent with the molecular basis of cognitive decline in AD patients. (6-month-old female ERα...) - / - In mice, ERα was almost not expressed, while the levels of ERβ and E2 were slightly increased but not statistically significant. ERβ expression was upregulated after administration of OST. Furthermore, ERα in 6-month-old mice... - / - In mice, the levels of the pro-apoptotic factor Bax were abnormally elevated, while the levels of the anti-apoptotic protein bcl-2 were decreased. The mechanism may be related to the activation of the BDNF-TrkB signaling pathway, increased phosphorylation of the PI3K / AKT pathway, and the blocking of the mitochondrial apoptosis cascade.

[47] .

[0288] In recent years, an increasing number of studies have demonstrated that OST (Osteoscopic Stem Cells) holds promise as a potential drug candidate for the treatment of Alzheimer's disease (AD): OST can promote the proliferation of endogenous neural stem cells. 48] Or by regulating endoplasmic reticulum stress [ 49] OST can improve AD-related pathological features through multiple mechanisms, including restoring estrogen-cholinergic system balance, neurotrophic factors, synaptic plasticity, and apoptosis. Our experimental results suggest that OST may improve ERα in 6-month-old females by restoring estrogen-cholinergic system balance, neurotrophic factors, synaptic plasticity, and apoptosis. - / - The learning and memory abilities of mice.

[0289] summary

[0290] OST treatment improved ERα in 6-month-old females - / - The mechanism by which the study of learning and memory dysfunction, anxiety-depression-like behavior, and pathological damage in mice may be through binding to ERs, reducing cholinergic damage, thereby enhancing synaptic plasticity, inhibiting apoptosis, and enhancing neurotransmitter expression, thus improving learning and memory abilities.

[0291] in conclusion

[0292] 1. OST effect on 6-month-old female peripheral ERs downregulated mice and ERα - / - The study showed improvement in both learning and memory abilities and pathological damage in mouse models.

[0293] 2. The mechanism by which OST improves learning and memory may be related to OST regulating the estrogen-cholinergic system, thereby increasing neuroplasticity, inhibiting neuronal apoptosis, and enhancing neurotransmitter function.

[0294] 3. OST on 6-month-old female ERα - / - The intervention in mouse models showed better results, possibly because the kidney-tonifying drug OST acts on relevant targets in peripheral target organs, thereby participating in the regulation of central cognitive function.

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Claims

1. Use of osthole in the preparation of a drug for protecting against cognitive impairment caused by estrogen receptor down-regulation.

2. Use according to claim 1, characterized in that, The osthole improves learning and memory ability under estrogen receptor down-regulation.

3. Use according to claim 2, characterized in that, The osthole improves learning and memory ability by regulating estrogen-cholinergic system to improve neural plasticity, inhibit neural apoptosis and enhance neurotransmitter.

4. Use according to claim 1, characterized in that, The osthole acts on peripheral target organs to participate in the regulation of central system cognitive function.

5. The use according to claim 1, characterized in that, The osthole improves pathological impairment under estrogen receptor down-regulation.

6. Use according to claim 1, characterized in that, The osthole relieves anxiety and depression under estrogen receptor down-regulation.

7. Use of osthole in the preparation of a drug for treating Alzheimer's disease.

8. Use according to claim 7, characterized in that, The Alzheimer's disease includes female Alzheimer's disease.

9. Use according to claim 8, characterized in that, The female Alzheimer's disease includes postmenopausal female Alzheimer's disease.

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