Application of eldecalcitol combined with risperidone in improving schizophrenia and preventing and treating risperidone side effect osteoporosis

Abstract

The application belongs to the technical field of medicine, and particularly relates to application of eldecalcitol combined with risperidone in improving schizophrenia and preventing and treating risperidone side effect osteoporosis. Specifically, the application proves that, by inhibiting hippocampal gamma-aminobutyric acid neuron iron death, the eldecalcitol restores brain neurotransmitter homeostasis, thereby improving schizophrenia in cooperation with risperidone; meanwhile, the eldecalcitol relieves risperidone-induced osteoporosis by regulating neural pathways and a direct pathway of preventing osteoblast iron death. Therefore, the application brings a new breakthrough to the treatment field of schizophrenia, and realizes the clinical effect of 'one arrow with two effects' by developing the combination of the eldecalcitol and the risperidone, so as to provide a new hope and direction for the treatment of schizophrenia patients, and therefore has a good practical application value.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to application of eldecalcitol combined with risperidone in improving schizophrenia and preventing and treating risperidone side effect osteoporosis. BACKGROUND

[0002] The information disclosed in this Background section is for the purpose of generally presenting the context of the application. The information disclosed in this Background section is not to be taken as an admission that it is prior art presented by nature of anticipation of any commonly owned application or otherwise known by anyone other than the inventor(s) before the filing date of this application.

[0003] Schizophrenia is a common severe mental disorder, which brings a huge burden to patients and society. It is a group of clinical syndromes composed of multiple symptoms, including positive symptoms (delusions and hallucinations), negative symptoms (blunted affect and diminished volition), cognitive impairment and emotional symptoms. At present, the widely used drug for treating schizophrenia in the clinic is the second-generation atypical antipsychotic drug represented by risperidone (RIS), which targets the dopamine (DA) and serotonin (5-HT) systems. It has been proved that RIS can partially alleviate the positive symptoms of schizophrenia, but it has weak efficacy in negative symptoms and cognitive dysfunction. Risperidone can partially block the binding of neurotransmitters by antagonizing the receptors, but its efficacy in fundamentally restoring the balance of neurotransmitters in the brain is limited, and high-risk doses have certain cytotoxicity, so its efficacy is limited.

[0004] A large amount of evidence shows that the hippocampus is a key area for regulating the dysfunction of patients with schizophrenia. The loss of hippocampal gamma-aminobutyric acid (GABA) interneurons will lead to the disinhibition of hippocampal pyramidal neurons, resulting in the over-activation of the hippocampus, and the hyperactivity of the hippocampus will affect the neurotransmitter homeostasis in the brain and induce the onset of schizophrenia. Considering that high-concentration risperidone has cytotoxicity, the hippocampal GABA neurons of schizophrenia patients receiving high-risk risperidone treatment are damaged, and it may further exacerbate the disturbance of neurotransmitter levels in the brain.

[0005] In addition, schizophrenia is closely related to osteoporosis. Besides genetic and environmental factors, the lack of gamma-aminobutyric acid levels may be the underlying regulatory mechanism. Studies have shown that central gamma-aminobutyric acid has an inhibitory effect on sympathetic nervous system excitability, and its lack will trigger excessive sympathetic nervous system, thereby inhibiting bone formation. In addition, the use of antipsychotic drugs can also produce other side effects, including osteoporosis, decreased bone density and increased risk of fractures. Studies have shown that risperidone-induced bone remodeling instability includes inhibition of osteogenic function and activation of osteoclastic function, which leads to bone loss through various pharmacological mechanisms, which can be summarized as neural pathways and direct pathways. In the neural pathway, risperidone induces hyperprolactinemia through its DA receptor antagonism and overactivates the sympathetic nervous system through its 5-HT receptor antagonism to indirectly induce osteoporosis. In addition, considering the possible down-regulation of RIS on gamma-aminobutyric acid and the sympathetic nervous system disinhibition triggered by its lack, this may exacerbate the activation of the sympathetic nervous system by RIS, thereby having a regulatory effect on osteogenic function, which may be another mechanism for risperidone to induce osteoporosis through neural pathways. At the same time, studies have shown that risperidone can directly affect the fate of bone cells, and risperidone affects the differentiation of osteoblasts by reducing autophagy flux and promoting apoptosis. Therefore, the neural pathway and direct pathway of risperidone leading to bone loss are worth further exploring.

[0006] Ferroptosis is a non-apoptotic form of necrotic cell death discovered in recent years, which is regulated and triggered by iron-dependent lipid peroxidation and regulated by multiple cellular metabolic pathways and multiple disease-related signal transduction pathways. The accumulation of reactive oxygen species (ROS) in cells is regulated by iron-dependent Fenton reaction and glutathione (GSH) loss, which is the direct cause of ferroptosis. Impaired glutathione peroxidase 4 (GPX4) activity and accumulation of redox-active iron are markers of ferroptosis. The entire ferroptosis process is usually accompanied by other phenotypic interactions, and usually involves autophagy abnormalities. Many pathological processes involved in ferroptosis, such as abnormal fatty acid and iron metabolism and ROS accumulation, are dependent on the excessive triggering and flux inhibition of autophagy. Therefore, the high-intensity activation of antipsychotic drugs to initiate autophagy and block autophagy flux to induce ferroptosis may be the source of its cytotoxicity. Studies have shown that DA receptor antagonists mediate autophagy-induced ferroptosis, and the excessive triggering of autophagy may be related to the pharmacological properties of the cationic amphiphilic drugs of antipsychotic drugs. This small molecule with the characteristics of a weakly basic hydrophobic base is also known as a lysosomal drug, which significantly accumulates in lysosomes through a mechanism called ion trapping. Lysosomal accumulation of lysosomal drugs and the resulting lysosomal membrane fluidization promote the dissociation of mTOR from the lysosomal membrane and inhibit the kinase activity of mTORC1, thereby inducing excessive activation of autophagy and possibly further triggering ferroptosis. Research reports that ferroptosis is closely related to schizophrenia and osteoporosis. These studies suggest that it is worth further exploring whether risperidone regulates hippocampal gamma-aminobutyric neurons and osteoblast function by directly inducing ferroptosis to affect the pathogenesis of schizophrenia and osteoporosis. SUMMARY

[0007] In view of the deficiencies in the prior art described above, the inventors have long-term technical and practical exploration, and provide the application of eldecalcitol combined with risperidone in improving schizophrenia and preventing and treating osteoporosis as a side effect of risperidone. The inventors have for the first time discovered and confirmed that the new active vitamin D analogue eldecalcitol (ED-71) improves schizophrenia and prevents and treats osteoporosis as a side effect of risperidone in synergy with risperidone (RIS), thereby exerting the effect of "one arrow with two effects". Based on the above research results, the present application is completed.

[0008] To achieve the above technical purpose, the technical scheme adopted by the present application is as follows:

[0009] In a first aspect, the present application provides the use of eldecalcitol in the preparation of a drug for promoting the therapeutic effect of risperidone on schizophrenia and preventing and treating its side effects.

[0010] Specifically, the promotion of the therapeutic effect of risperidone on schizophrenia is manifested by improving the level of gamma-aminobutyric acid in hippocampal neurons induced by risperidone to recover the level of gamma-aminobutyric acid, thereby synergizing with risperidone to relieve the symptoms of schizophrenia.

[0011] The side effects of risperidone include bone loss mediated by it, and further related diseases such as osteoporosis. More specifically, the elocalcitol alleviates the risperidone-induced osteoporosis by regulating the neural pathway and the direct pathway of preventing osteoblast iron death.

[0012] In a second aspect of the present application, there is provided a use of elocalcitol in combination with risperidone in the preparation of a drug for treating schizophrenia.

[0013] In a third aspect of the present application, there is provided a pharmaceutical composition for treating schizophrenia, wherein the active ingredients of the pharmaceutical composition at least comprise elocalcitol and risperidone.

[0014] When the elocalcitol is used in combination with risperidone, the mass ratio of the two is 1-100x10 -6 : 1; preferably 5x10 -5 : 1.

[0015] In a fourth aspect of the present application, there is provided a method for treating schizophrenia, which comprises administering a therapeutically effective dose of the above-mentioned pharmaceutical composition to a subject.

[0016] Compared with the prior art, the above one or more technical solutions have the following beneficial effects:

[0017] The above technical solution firstly proposes a new active vitamin D analogue elocalcitol to improve schizophrenia in cooperation with risperidone and prevent the side effect of osteoporosis, thereby playing a "one arrow two" effect, which shows that it has the prospect of developing into a drug for treating schizophrenia, opens up a new drug use for elocalcitol, and lays an experimental foundation and provides a new perspective for developing a new drug for treating schizophrenia with better therapeutic effect and lower side effect, thus having important clinical significance and social benefits. BRIEF DESCRIPTION OF DRAWINGS

[0018] The drawings constituting a part of the specification illustrate the present application and, together with the description, serve to explain the principles of the present application. They should not be considered as limiting the present application in any way.

[0019] Figure 1This example illustrates how idecalcitol, in an embodiment of the present invention, improves risperidone-induced ferroptosis of GABAergic neurons in the hippocampus and restores GABA levels, thereby synergizing with risperidone to alleviate schizophrenia symptoms. (A) Open field test route. (B) Statistical analysis of total movement distance, average movement speed, and total stagnation time. (C) Three-chamber social test route. (D) Statistical analysis of social cognition ability. (E) Line graph of water maze latency. (F) Water maze test route. (G) Statistical analysis of platform crossings and platform dwell time during the water maze test. Data are presented as mean ± standard deviation (n = 6). (H) Hippocampal HE staining and Nissl staining. (I) Statistical analysis of GABA levels in mouse hippocampus. Data are presented as mean ± standard deviation (n = 3). (J) Transmission electron microscopy image of mouse hippocampal neuronal cell line HT22 cells. (K) Fluorescence image of HT22 cells co-stained with a mitochondrial probe and a mitochondrial ferrous ion probe. (L) Fluorescence image of lipid peroxidation probe in HT22 cells;

[0020] Figure 2 This example shows that eldecalcitol improves osteoporosis in risperidone-treated schizophrenia mice via neural pathways and a direct pathway that inhibits osteoblast ferroptosis. (A) Typical three-dimensional and two-dimensional structural images of the femur obtained using Micro-CT. (B) HE and Masson staining images. (C) Immunohistochemical staining images of RUNX2, ALP, TRAP, and CTSK. (D) Statistical analysis of prolactin levels in mouse femurs. (E) Statistical analysis of norepinephrine levels in mouse femurs. Data are expressed as mean ± standard deviation (n = 3). (F) ALP and Alizarin Red staining images of MC3T3-E1 cells. (G) Transmission electron microscopy images of MC3T3-E1 cells. (H) Fluorescence image of MC3T3-E1 cells co-stained with a mitochondrial probe and a mitochondrial ferrous ion probe. (I) Fluorescence image of MC3T3-E1 cells with a lipid peroxidation probe. DETAILED DESCRIPTION

[0021] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0022] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0023] In one embodiment of the present application, the use of alfacalcidol in the preparation of a medicament for promoting the therapeutic effect of risperidone on schizophrenia while preventing and treating the side effects of risperidone is provided.

[0024] The promotion of the therapeutic effect of risperidone on schizophrenia is specifically manifested by improving the level of gamma-aminobutyric acid by recovering hippocampal neuron iron death induced by risperidone, thereby relieving the symptoms of schizophrenia in cooperation with risperidone.

[0025] The side effects of risperidone include bone loss mediated by risperidone, and further related diseases such as osteoporosis. More specifically, the alfacalcidol relieves risperidone-induced osteoporosis by regulating the neural pathway and directly preventing osteoblast iron death.

[0026] In another embodiment of the present application, the use of alfacalcidol in combination with risperidone in the preparation of a medicament for treating schizophrenia is provided.

[0027] When the alfacalcidol is used in combination with risperidone, the mass ratio of the two is 1-100×10 -6 : 1; preferably 5×10 -5 : 1.

[0028] The treatment of schizophrenia is specifically manifested by the use of alfacalcidol in combination with risperidone to treat schizophrenia, while alfacalcidol is used to prevent and treat the side effects mediated by risperidone.

[0029] Further, the side effects of risperidone include bone loss mediated by risperidone, and further related diseases such as osteoporosis. More specifically, the alfacalcidol relieves risperidone-induced osteoporosis by regulating the neural pathway and directly preventing osteoblast iron death.

[0030] In another embodiment of the present application, a pharmaceutical composition for treating schizophrenia is provided, wherein the active ingredients of the pharmaceutical composition at least include alfacalcidol and risperidone.

[0031] When the alfacalcidol is used in combination with risperidone, the mass ratio of the two is 1-100×10 -6 : 1; preferably 5×10 -5 : 1.

[0032] Further, the pharmaceutical composition further comprises at least one non-pharmaceutically active ingredient.

[0033] The non-pharmaceutically active ingredients can be carriers, excipients, diluents, and the like, which are commonly used in the pharmaceutical field. Also, the dosage form can be prepared into a powder, granule, suspension, emulsion, syrup, spray, and the like, for oral administration, external use, suppository, and sterile injection solution, according to the conventional method.

[0034] The non-pharmaceutically active ingredients, such as carriers, excipients, and diluents, which can be included, are well known in the field, and one of ordinary skill in the art can determine whether they meet the clinical standards.

[0035] In another embodiment of the present application, the carriers, excipients, and diluents include, but are not limited to, lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil, and the like.

[0036] In another embodiment of the present application, the pharmaceutical composition of the present application can be administered into the body by known methods. For example, it can be delivered into the body by intravenous systemic delivery or local injection into the tissue of interest. Such administration can be performed via a single dose or multiple doses. One of ordinary skill in the art understands that the actual dose to be administered in the present application can vary greatly depending on various factors, such as the target cell, the type of organism or its tissue, the general condition of the subject to be treated, the route of administration, the mode of administration, and the like.

[0037] In another embodiment of the present application, the subject of the pharmaceutical administration can be a human and a non-human mammal, such as a mouse, a rat, a guinea pig, a rabbit, a dog, a monkey, a chimpanzee, and the like.

[0038] In another embodiment of the present application, there is provided a method of treating schizophrenia, the method comprising administering to a subject a therapeutically effective dose of the above pharmaceutical composition.

[0039] The subject refers to an animal, preferably a mammal, and most preferably a human, who has been the object of treatment, observation, or experiment.

[0040] The term "therapeutically effective amount" means an amount of the active compound or pharmaceutical agent, including the compounds of the present application, which elicits the biological or medicinal response that is being sought in a tissue system, animal or human by a researcher, veterinarian, medical doctor or other medical person, including alleviation or partial alleviation of the symptoms of the disease, syndrome, condition, or disorder being treated. It must be appreciated that the optimal dosage and spacing of administration of the active ingredients described herein are determined by their properties, and external conditions such as the form, route and site of administration, and the particular mammal being treated, and such optimal dosages can be determined using routine techniques. It must also be appreciated that the optimal course of treatment, i.e., the daily dosage of the compound over a given period of time, can be determined using methods known in the art.

[0041] The present application is further illustrated by the following examples, which are not intended to limit the scope of the application. Any simple modification, equivalent variation and modification made to the embodiments according to the technical essence of the present application shall fall within the scope of the technical solutions of the present application.

[0042] Examples

[0043] Materials and Methods

[0044] 1. Establishment of animal model of schizophrenia

[0045] C57BL / 6J mice were used, 10 mice per group in a total of four groups, including a control group, a schizophrenia group, a risperidone monotherapy group and an alfacalcidol combined with risperidone treatment group. The control group of mice was injected intraperitoneally with phosphate buffered saline (PBS) daily, and was given intragastrically with glycerol. The other three groups of mice were all injected intraperitoneally with dizocilpine (MK-801) at a dose of 1 mg / kg / day for 14 consecutive days to successfully construct a schizophrenia model. Subsequently, MK-801 was continuously given in the same way until the end of the modeling process to maintain the performance of psychotic symptoms. The risperidone monotherapy group was injected intraperitoneally with risperidone at a dose of 1 mg / kg / day for 8 weeks after the successful establishment of the schizophrenia model, while being given intragastrically with glycerol. The alfacalcidol combined with risperidone treatment group was injected intraperitoneally with risperidone at a dose of 1 mg / kg / day and was given intragastrically with alfacalcidol at a dose of 50 ng / kg / day for 8 weeks after the successful establishment of the SZ model was verified.

[0046] 2. Cell culture

[0047] The hippocampal neuron cell line HT22 cells were cultured in high-glucose DMEM medium containing 10% fetal bovine serum and 1% antibiotics (penicillin, streptomycin). The osteoblast cell line MC3T3-E1 cells were cultured in a-MEM medium containing 10% fetal bovine serum and 1% antibiotics (penicillin, streptomycin). The cells were incubated at 37°C in a humidified environment with 5% CO2.

[0048] 3. Open field test

[0049] The open field test apparatus is a box made of black high-density polyethylene plates that are fastened together and placed on a plastic floor. The behavior of the mice is recorded with a camera mounted above the open field. The total distance moved, the total time of immobility, and the average speed of movement of the test mice are detected and statistically analyzed.

[0050] 4. Three-chamber social interaction test

[0051] The test mice are first placed in an empty apparatus (i.e., a three-chamber box consisting of three interconnected lined compartments) and allowed to habituate to the setting. On the test day, the test mice are habituated to the central compartment with the door closed for 5 minutes. After the habituation phase, the test mice are trained and tested to assess their social cognitive ability. The training phase allows the test mice to freely explore the compartment with a conspecific mouse or another empty compartment for 10 minutes. The test phase, which is performed 10 minutes later, allows the test mice to interact with a known mouse (the same mouse as in the training phase) and a stranger mouse in the other outer compartment, and the test mice are tested for their preference of interaction for 10 minutes. The time of close contact of the test mice with the known mouse and the stranger mouse is detected and statistically analyzed.

[0052] 5. Morris water maze test

[0053] The animals are tested in a circular pool located in the maze room. The platform is submerged 1.5 cm below the water surface and maintained at 23-25°C throughout the test. Non-toxic titanium dioxide is added to make the water opaque and hide the platform. The experiment is divided into two phases, including a learning phase and a test phase. In the learning phase, the animals are placed in the pool and must find the hidden platform underwater by swimming. The location of the hidden platform remains the same for all mice, and the starting position is randomly selected. Each mouse has 60 seconds to find the hidden platform, and the latency to find the platform is recorded. If the animal fails to reach the hidden platform within 60 seconds, the mouse is allowed to explore the maze cues for 10 seconds on the platform by gently pushing the mouse forward from the root of their tail and guiding them to the target location. In the test phase, the hidden platform is removed, and the mouse is allowed to freely explore for 60 seconds, and the number of crossings and the time spent on the platform are statistically analyzed.

[0054] 6. Micro-CT

[0055] After the modeling is completed, the femur is aseptically dissected for Micro-CT scanning. Subsequently, these samples are scanned using a Micro-CT analysis system, and three-dimensional images are reconstructed at a resolution of 10 microns.

[0056] 7. HE staining

[0057] HE staining to observe histological changes. After deparaffinization and hydration, the sections were immersed in hematoxylin staining solution for 15 minutes, washed in distilled water, immersed in eosin for 7 minutes, washed again in distilled water, and finally dehydrated and mounted. Thereafter, the sections were observed and images were obtained under an optical microscope.

[0058] 8. Nissl staining

[0059] After deparaffinization and hydration of the sections, Nissl staining solution was added dropwise to the sections on the slides, and a coverslip was added, staining for 10 minutes. After the staining was complete, the slides were thoroughly rinsed with distilled water to remove unbound dye, ensuring a clear field of view, and then the rinsed slides were mounted with gum, and images were obtained under an optical microscope.

[0060] 9. Immunohistochemical staining and TRAP staining

[0061] After deparaffinization and hydration of the paraffin sections, the sections were pretreated with 0.3% hydrogen peroxide for 30 min, and then blocked with 1% bovine serum albumin (BSA) / PBS for 30 min at room temperature. Then the sections were incubated with the corresponding antibodies overnight. The next day, after rewarming, they were rinsed in PBS, and then the sections were subjected to secondary antibody staining for 1 hour at room temperature. Subsequently, DAB developing solution was prepared, added dropwise to the sections, and observed using an optical microscope and stopped in time. After the sections stained by ALP & TRAP double staining were developed, they were rinsed in distilled water, and then TRAP staining solution was added dropwise, and reacted for 15 minutes at room temperature until red staining was observed. Finally, all the sections were counterstained with methyl green stain, and then images were taken under an optical microscope.

[0062] 10. Enzyme-linked immunosorbent assay (ELISA)

[0063] Sample preparation. For homogenization, the tissue was taken and its mass was weighed. 1 mL of 0.9% saline was added per 200 mg, and after thorough grinding, the tissue homogenate was transferred to an EP tube, which was centrifuged at 12000 rpm for 10 min at 4°C. The supernatant was used for ELISA detection. The levels of prolactin, norepinephrine, and gamma-aminobutyric acid in the sample were detected using the relevant kit, and the ELISA results were evaluated using an enzyme labeler.

[0064] 11. Transmission electron microscopy

[0065] The ultrastructure of HT22 and MC3T3-E1 cells was observed using transmission electron microscopy. Cells were fixed in 2.5% glutaraldehyde at 4°C for 2 h, then postfixed in 1% ozone tetroxide at 4°C for 1 h, dehydrated, and embedded in epoxy resin. Tissues were cut into thin sections using an ultramicrotome (EMUC7, Leica). Ultrathin sections (60–80 nm) mounted on copper grids were observed using a Thermo Fisher Talos F200C transmission electron microscope and compared with 8% uranyl acetate and lead citrate.

[0066] 12. Mitochondrial ferrous ion detection

[0067] Mito-FerroGreen is a special cell dye used to detect ferrous ions (Fe) in mitochondria. 2+ In order to detect the Fe 2+ The cells were seeded on a confocal microplate and incubated with Mito-FerroGreen (5 μM), Mito-Tracker (50 nM) and nuclear staining dye Hoechst 33342 (100 nM) at 37°C for 30 min. The cells were then imaged on a confocal microscope.

[0068] 13.BODIPY 581 / 591C11

[0069] To assess lipid-ROS levels, cells were seeded onto confocal microplates and stained with 5 μM of the lipid peroxidation sensor BODIPY 581 / 591C11 for 30 minutes at 37°C. The cells were then replaced with PBS and observed on a confocal microscope.

[0070] 14. Alkaline phosphatase (ALP) staining and Alizarin red (ARS) staining

[0071] After culture, cells were fixed with 4% paraformaldehyde for 15 minutes and stained with alkaline phosphatase to detect osteoblast alkaline phosphatase activity. The degree of osteoblast mineralization was assessed by fixing cells with 4% paraformaldehyde for 15 minutes and then staining with 1% AR staining solution. Images were acquired using an optical microscope (CKX-41, Olympus Corporation, Japan).

[0072] result:

[0073] 1. Idecalcitol improves risperidone-induced hippocampal neuronal ferroptosis and restores γ-aminobutyric acid levels, thereby synergizing with risperidone to alleviate schizophrenia symptoms.

[0074] To determine whether idecalcitol could synergize with risperidone to attenuate MK-801-induced positive symptoms of schizophrenia, we measured spontaneous locomotor behavior in an open field box ( Figure 1A) The total distance and average speed of mice in the MK-801 group were significantly increased, and the total immobility time was significantly shortened compared with the control group. Risperidone administration improved this hyperactive behavior, and the combination of alfacalcidol further reversed the hyperactive behavior induced by MK-801. Figure 1 B) These results show that alfacalcidol cooperates with risperidone to improve the hyperactivity induced by MK-801.

[0075] We evaluated the effect of alfacalcidol combined with risperidone on the social cognitive ability of mice by a three-chamber social test to explore its efficacy on the negative symptoms of schizophrenia. Figure 1 C) In the social preference test, the MK-801 group spent significantly less time exploring the chamber with a new mouse compared with the familiar chamber compared with the control group. There was no significant difference between the risperidone group and the MK-801 group. Alfacalcidol combined with risperidone significantly alleviated the social avoidance induced by MK-801. Figure 1 D) These results show that alfacalcidol combined with risperidone effectively reversed the negative symptoms induced by MK-801.

[0076] During the training process of the water maze test, the latency of the MK-801 group to find the platform was longer than that of the control group. Risperidone did not improve the latency of the MK-801 group, but alfacalcidol combined with risperidone shortened the latency of schizophrenic mice to find the platform. Figure 1 E-F) In addition, during the test, there was no significant difference in the number of times across the platform and the platform residence time between the MK-801 group and the risperidone group, but the combination of alfacalcidol improved the cognitive ability of schizophrenic mice. Figure 1 G)

[0077] The hippocampus is an important brain region that regulates schizophrenia. Gamma-aminobutyric acid neurons in the hippocampus regulate the balance of neurotransmitters in the brain by secreting gamma-aminobutyric acid, thereby affecting the occurrence and development of various mental symptoms. In our study, Nissl staining was used to assess the viability and function of hippocampal neurons. The viability of hippocampal neurons in the MK-801 + risperidone group was lower than that in the MK-801 group alone, and alfacalcidol partially rescued this damage. Figure 1 H)

[0078] Further detection of the effect of hippocampal neuron damage on the instability of neurotransmitters in the brain, ELISA method was used to detect the level of neurotransmitters in the brain homogenate of mice. The results showed that compared with the MK-801 group, the level of inhibitory neurotransmitter gamma-aminobutyric acid in the hippocampus of the risperidone group was lower, and the combination of alfacalcidol alleviated the loss of gamma-aminobutyric acid content in the hippocampus. Figure 1 I)

[0079] To explore the specific mechanism of hippocampal GABA reduction, we selected the hippocampal neuron cell line HT22 for in vitro experiments. This cell line can synthesize and secrete GABA, which can meet our research purposes. To explore the reason for the decrease in cell activity under the action of risperidone, we used transmission electron microscopy to observe the ultrastructure of each group. The results showed that compared with the MK-801 group alone, the HT22 cell mitochondria were shortened, vacuolated, and accompanied by increased mitochondrial electron density, showing typical characteristics of ferroptosis. Pre-treatment with alfacalcidol significantly alleviated the adverse ultrastructure of cell mitochondria Figure 1 J).

[0080] To verify the hippocampal neuron ferroptosis induced by risperidone and the functional role of alfacalcidol in hippocampal neuron damage, we co-stained HT22 cells with mitochondrial probe Mito-Tracker and mitochondrial ferrous ion probe Mito-FerroGreen, and further used BODIPY 581 / 591C11 to detect lipid peroxidation levels. The results showed that compared with the MK-801 group, risperidone significantly exacerbated iron accumulation in HT22 cell mitochondria and cell lipid peroxidation, and these ferroptosis characteristics were alleviated by alfacalcidol pre-treatment Figure 1 K-L).

[0081] 2. Alfacalcidol improves osteoporosis in risperidone-treated schizophrenic mice through the neural pathway and direct pathway of inhibiting osteocyte ferroptosis

[0082] In addition to psychiatric symptoms, we further explored the effects of risperidone and alfacalcidol on bone remodeling in MK-801-induced schizophrenic mice. Micro-CT showed that MK-801-induced schizophrenic mice had reduced bone mass compared to the CON group, and the bone mass of schizophrenic mice treated with risperidone was further reduced, while the bone mass of mice given pre-treatment with alfacalcidol was significantly restored. Compared with risperidone group mice, alfacalcidol group had increased trabecular bone number and thickness Figure 2 A), and further HE and Masson staining showed similar results Figure 2 B). To clarify the bone remodeling regulation mechanism of alfacalcidol in restoring bone mass, we used immunohistochemical staining to detect bone formation and bone resorption-related factors. As expected, immunohistochemical results showed that risperidone administration reduced the expression levels of osteoblast-related factors RUNX2 and ALP in schizophrenic mice, while combined with alfacalcidol significantly promoted bone formation in MK-801+risperidone group mice. In addition, risperidone promoted bone resorption in schizophrenic mice, while alfacalcidol inhibited the excessive activation of mouse bone resorption levels Figure 2C). Therefore, prophylactic administration of alfacalcidol can significantly increase bone formation in MK-801 + risperidone group mice to partially restore bone mass.

[0083] To clarify the mechanism of risperidone-induced osteoporosis in schizophrenic mice and the regulatory effect of alfacalcidol, we explored the neural pathway and direct effect. First, the effect of neural pathway on bone metabolism, studies have shown that the level of gamma-aminobutyric acid plays an important role in the regulation of bone formation, considering the difference in hippocampal gamma-aminobutyric acid content between groups Figure 1 I), therefore, alfacalcidol administration or by improving central gamma-aminobutyric acid to promote osteogenesis in MK-801 + risperidone group mice. In addition, studies have shown that risperidone can regulate neural circuits by antagonizing DA receptors and 5-HT receptors to induce hyperprolactinemia and sympathetic nerve excitement to induce bone metabolism instability. We detected the levels of prolactin and norepinephrine in femoral tissue homogenate by ELISA, and found that alfacalcidol down-regulated the risperidone-induced high prolactin level and high norepinephrine level Figure 2 D-E).

[0084] In addition, to explore the direct effect of alfacalcidol on bone formation in risperidone-treated schizophrenic mice, we selected the MC3T3-E1 cell line for in vitro experiments. We induced MC3T3-E1 osteogenic differentiation, and used ALP staining and alizarin red staining to detect osteogenic differentiation and mineralization function, and the results showed that ED-71 pretreatment partially alleviated the osteoblast dysfunction induced by risperidone, and increased the formation of mineralized nodules Figure 2 F). To clarify the mechanism of drug effects on cell function, we observed the ultrastructure of each group by transmission electron microscopy, and found that compared with the MK-801 group alone, the mitochondria of osteoblasts were shortened, vacuolated, and accompanied by increased mitochondrial electron density, showing the typical characteristics of ferroptosis, and alfacalcidol pretreatment significantly alleviated the adverse ultrastructure of cell mitochondria Figure 2 G).

[0085] To verify the ferroptosis of osteoblasts induced by risperidone and the functional role of alfacalcidol in osteoblast damage, we co-stained MC3T3-E1 cells with mitochondrial probe Mito-Tracker and mitochondrial ferrous ion probe Mito-FerroGreen, and further detected lipid peroxidation levels with BODIPY 581 / 591C11. The results showed that compared with the MK-801 group, risperidone significantly aggravated the accumulation of iron in the mitochondria of MC3T3-E1 cells and cell lipid peroxidation, and this ferroptosis characteristic could be alleviated by alfacalcidol pretreatment Figure 2 H-I).

[0086] In summary, alfacalcidol improves schizophrenia by inhibiting hippocampal GABAergic neuron ferroptosis to restore neurotransmitter homeostasis in the brain in conjunction with risperidone; at the same time, alfacalcidol relieves risperidone-induced osteoporosis by regulating neural pathways and directly preventing osteoblast ferroptosis. Therefore, this study is expected to bring new breakthroughs to the field of schizophrenia treatment, and to achieve the clinical effect of "one arrow with two effects" by developing alfacalcidol in conjunction with risperidone, in order to provide new hope and direction for the treatment of patients with schizophrenia.

[0087] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Those skilled in the art can make various modifications and changes to the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. Use of eldecalcitol in the manufacture of a medicament for promoting the therapeutic effect of risperidone on schizophrenia while preventing the side effects of risperidone. The promotion of the therapeutic effect of risperidone on schizophrenia is manifested by the improvement of GABA levels by improving hippocampal GABA neuron ferroptosis induced by risperidone, thereby alleviating the symptoms of schizophrenia in combination with risperidone. The side effects of risperidone include bone loss mediated by risperidone, and further induced osteoporosis.

2. The use according to claim 1, characterized in that, The prevention of side effects of risperidone by eldecalcitol is manifested by alleviating risperidone-induced osteoporosis through regulating neural pathways and directly preventing osteoblast ferroptosis.

3. Use of eldecalcitol in combination with risperidone in the preparation of a drug for treating schizophrenia; when the eldecalcitol is used in combination with risperidone, the mass ratio of the two is 1-100 x 10 -6 :

1. The schizophrenia treatment is manifested by the combination of eldecalcitol and risperidone in the synergistic treatment of schizophrenia, while eldecalcitol is used to prevent the side effects of risperidone, including bone loss mediated by risperidone, and further induced osteoporosis.

4. The use according to claim 3, wherein the compound is ###0002### The combination of eldecalcitol and risperidone in the synergistic treatment of schizophrenia is manifested by the improvement of GABA levels by improving hippocampal neuron ferroptosis induced by risperidone, thereby alleviating the symptoms of schizophrenia in combination with risperidone.

5. The use according to claim 3, wherein the compound is ###0002### The prevention of side effects of risperidone by eldecalcitol is manifested by alleviating risperidone-induced osteoporosis through regulating neural pathways and directly preventing osteoblast ferroptosis.

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