Application of eldecalciferol and risperidone in improvement of schizophrenia and prevention and treatment of osteoporosis caused by risperidone side effect

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of idedecalciferol and risperidone in improvement of schizophrenia and prevention and treatment of osteoporosis caused by risperidone side effects. Specifically, research proves that idedecalciferol recovers neurotransmitter homeostasis in the brain by inhibiting hippocampus gamma-aminobutyric acid ergic neuron ferroptosis, so that idedecalciferol cooperates with risperidone to improve schizophrenia; meanwhile, the eldecalciferol relieves the osteoporosis induced by risperidone through a direct way of regulating and controlling a nerve way and preventing osteoblast ferroptosis. Therefore, a new breakthrough is brought to the field of schizophrenia treatment, the clinical curative effect of'one arrow and two sculptures' is achieved by developing the combination of the idedecalciferol and the risperidone, new hope and direction are expected to be provided for treatment of schizophrenia patients, and therefore the application has good practical application value.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to the application of idecalcitol combined with risperidone in improving schizophrenia while preventing and treating osteoporosis, a side effect of risperidone. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Schizophrenia is a common severe mental disorder that brings a huge burden to patients and society. It is a clinical syndrome composed of a variety of symptoms, including positive symptoms (delusions and hallucinations), negative symptoms (affective blunting and avolition), cognitive impairment and emotional symptoms. At present, the drugs widely used in the clinic for the treatment of schizophrenia are second-generation atypical antipsychotics represented by risperidone (RIS), which target the dopamine (DA) system and the 5-hydroxytryptamine (5-HT) system and have been shown to partially relieve the positive symptoms of schizophrenia, but have weak efficacy in negative symptoms and cognitive dysfunction. Although risperidone can partially block the binding of transmitters by antagonizing transmitter receptors, it has limited efficacy in fundamentally restoring the balance of transmitters in the brain, 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 functional disorders in patients with schizophrenia. The loss of hippocampal γ-aminobutyric acid (GABA) interneurons will lead to the disinhibition of hippocampal pyramidal neurons, resulting in excessive activation of the hippocampus. The hyperactivity of the hippocampus will affect the homeostasis of neurotransmitters such as DA and 5-HT in the brain and induce the onset of schizophrenia. Considering that high concentrations of risperidone are cytotoxic, the hippocampal γ-aminobutyric acid neurons of schizophrenia patients receiving high-risk risperidone treatment may be damaged, and may further aggravate the disorder of neurotransmitter levels in the brain.

[0005] In addition, schizophrenia is closely related to osteoporosis. In addition to genetic and environmental factors, the lack of γ-aminobutyric acid levels may be a potential regulatory mechanism. Studies have shown that central γ-aminobutyric acid has the effect of inhibiting the excitability of the sympathetic nervous system, and its deficiency will cause excessive excitation of the sympathetic nerves, 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 osteoblastic function and activation of osteoclast function, which leads to bone loss through a variety of 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 downregulation of RIS on γ-aminobutyric acid and the sympathetic disinhibition caused by its deficiency, this may aggravate the activation of the sympathetic nervous system by RIS, thereby regulating the osteogenic function, which may be another mechanism by which risperidone induces osteoporosis through neural pathways. At the same time, studies have shown that risperidone can directly affect the fate of cells in the bone. Risperidone affects the differentiation of osteoblasts by reducing autophagic flux and promoting cell apoptosis. Therefore, the neural and direct pathways of risperidone-induced bone loss deserve further exploration.

[0006] Ferroptosis is a non-apoptotic necrotic cell death form discovered in recent years. It is regulated and triggered by iron-dependent lipid peroxidation and is 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 the iron-dependent Fenton reaction and glutathione (GSH) loss, which is the direct cause of ferroptosis. Impaired activity of glutathione peroxidase 4 (GPX4) and accumulation of redox-active iron are hallmarks 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, rely on excessive triggering of autophagy and flux inhibition. Therefore, the high intensity activation of autophagy initiation and blocking of autophagic flux by antipsychotic drugs to induce ferroptosis may be the source of their cytotoxicity. Studies have shown that DA receptor antagonists mediate autophagy-induced ferroptosis, and excessive triggering of autophagy may be related to the pharmacological properties of cationic amphiphilic drugs of antipsychotic drugs. This small molecule with hydrophobic weak base properties is also known as a lysosomal drug, which accumulates significantly in lysosomes through a mechanism called ion capture. Lysosomal accumulation of lysosomal drugs and the subsequent 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 may further trigger ferroptosis. Research reports that ferroptosis is closely related to schizophrenia and osteoporosis. These studies suggest that whether risperidone regulates the function of hippocampal γ-aminobutyric acid neurons and osteoblasts by directly inducing ferroptosis and thus affects the onset of schizophrenia and osteoporosis deserves further exploration. Summary of the invention

[0007] In view of the deficiencies in the above-mentioned prior art, the inventors, after long-term technical and practical exploration, provide the use of idecalcitol combined with risperidone in improving schizophrenia while preventing and treating osteoporosis, a side effect of risperidone. The present invention first discovered and confirmed through research that the new active vitamin D analog idecalcitol (ED-71) cooperates with risperidone (RIS) to improve schizophrenia while preventing and treating osteoporosis, a side effect of risperidone, thereby achieving the effect of "killing two birds with one stone". Based on the above research results, the present invention is completed.

[0008] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0009] The first aspect of the present invention provides the use of idecalcitol in the preparation of a drug for promoting the therapeutic effect of risperidone on schizophrenia while preventing and treating its side effects.

[0010] The promoting effect of risperidone on the treatment of schizophrenia is specifically manifested in that the γ-aminobutyric acid level is restored by improving risperidone-induced ferroptosis of hippocampal neurons, thereby cooperating with risperidone to alleviate schizophrenia symptoms.

[0011] The side effects of risperidone include bone loss mediated by risperidone, which in turn induces related diseases such as osteoporosis. More specifically, idecalcitol alleviates risperidone-induced osteoporosis by regulating neural pathways and preventing direct pathways of osteoblast ferroptosis.

[0012] The second aspect of the present invention provides the use of idecalcitol combined with risperidone in the preparation of a drug for treating schizophrenia.

[0013] The third aspect of the present invention provides a pharmaceutical composition for treating schizophrenia, wherein the active ingredients of the pharmaceutical composition at least include idecalcitol and risperidone.

[0014] When the idecalcitol and risperidone are used together, the mass ratio of the two is 1-100×10 -6 : 1; preferably 5×10 -5 :1.

[0015] A fourth aspect of the present invention provides a method for treating schizophrenia, comprising administering a therapeutically effective dose of the above pharmaceutical composition to a subject.

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

[0017] The above technical scheme proposes for the first time that a new active vitamin D analog idecalcitol can cooperate with risperidone to improve schizophrenia while preventing its side effect osteoporosis, thereby achieving the therapeutic effect of "killing two birds with one stone", indicating that it has the prospect of being developed into a drug for the treatment of schizophrenia, opening up new drug uses for idecalcitol, and also laying an experimental foundation and providing a new perspective for the development of new drugs for the treatment of schizophrenia with better therapeutic effects and lower side effects. Therefore, it has important clinical significance and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0019] Figure 1In the present invention, idecalcitol improves risperidone-induced hippocampal γ-aminobutyric acid neuronal ferroptosis and restores γ-aminobutyric acid levels, thereby cooperating with risperidone to relieve schizophrenia symptoms. (A) Open field test route map. (B) Statistical analysis of total moving distance, average moving speed and total stagnation time. (C) Three-box social experiment route map. (D) Statistical analysis of social cognitive ability. (E) Water maze latency line graph. (F) Water maze experiment route map. (G) Statistical analysis of the number of platform crossings and platform residence time during the water maze test. The above data are expressed as mean ± standard deviation (n = 6). (H) Hippocampal HE staining and Nissl staining. (I) Statistical analysis of mouse hippocampal γ-aminobutyric acid levels. Data are expressed as mean ± standard deviation (n = 3). (J) Transmission electron microscopy image of mouse hippocampal neuronal cell line HT22 cells. (K) Fluorescence image of HT22 cell mitochondrial probe co-stained with mitochondrial ferrous ion probe. (L) Fluorescence image of lipid peroxidation probe in HT22 cells;

[0020] Figure 2 The present invention shows that idecalcitol improves osteoporosis in risperidone-administered schizophrenia mice through neural pathways and direct pathways that inhibit osteoblast ferroptosis. (A) Typical femoral three-dimensional and two-dimensional structural images were obtained using Micro-CT. (B) HE staining and Masson staining images. (C) RUNX2, ALP&TRAP and CTSK immunohistochemical staining images. (D) Statistical analysis of prolactin levels in mouse femurs. (E) Statistical analysis of norepinephrine in mouse femurs. The above data are expressed as mean ± standard deviation (n = 3). (F) MC3T3-E1 cell ALP and Alizarin Red staining images. (G) MC3T3-E1 cell transmission electron microscopy images. (H) Fluorescence images of MC3T3-E1 cell mitochondrial probe and mitochondrial ferrous ion probe co-stained. (I) Fluorescence images of MC3T3-E1 cell lipid peroxidation probe. DETAILED DESCRIPTION

[0021] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present 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, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0023] In a typical embodiment of the present invention, there is provided a use of idecalcitol in the preparation of a drug for promoting the therapeutic effect of risperidone on schizophrenia while preventing and treating its side effects.

[0024] The promoting effect of risperidone on the treatment of schizophrenia is specifically manifested in that the γ-aminobutyric acid level is restored by improving risperidone-induced ferroptosis of hippocampal neurons, thereby cooperating with risperidone to alleviate schizophrenia symptoms.

[0025] The side effects of risperidone include bone loss mediated by risperidone, which in turn induces related diseases such as osteoporosis. More specifically, idecalcitol alleviates risperidone-induced osteoporosis by regulating neural pathways and preventing direct pathways of osteoblast ferroptosis.

[0026] In another specific embodiment of the present invention, there is provided use of idecalcitol combined with risperidone in the preparation of a drug for treating schizophrenia.

[0027] Wherein, when the idecalcitol and risperidone are used in combination, 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 as follows: while idecalcitol is combined with risperidone to synergistically treat schizophrenia, idecalcitol is used to prevent and treat the side effects mediated by risperidone.

[0029] Furthermore, the side effects of risperidone include bone loss mediated by risperidone, which in turn induces related diseases such as osteoporosis. More specifically, idecalcitol alleviates risperidone-induced osteoporosis by regulating neural pathways and preventing direct pathways of osteoblast ferroptosis.

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

[0031] When the idecalcitol and risperidone are used together, the mass ratio of the two is 1-100×10 -6 : 1; preferably 5×10 -5 :1.

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

[0033] The inactive ingredients of the drug can be carriers, excipients and diluents commonly used in pharmacy, etc. Moreover, according to the usual method, it can be made into oral preparations, external preparations, suppositories and sterile injection solutions in the form of powders, granules, suspensions, emulsions, syrups, sprays, etc.

[0034] The non-drug active ingredients such as carriers, excipients and diluents that may be included are well known in the art, and those skilled in the art can determine whether they meet clinical standards.

[0035] In another specific embodiment of the present invention, the carrier, excipient and diluent include but are not limited to lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

[0036] In another specific embodiment of the present invention, the medicine of the present invention can be administered to the body by known means. For example, it can be delivered to the tissue of interest by intravenous systemic delivery or local injection. Such administration can be carried out via a single dose or multiple doses. It is understood by those skilled in the art that the actual dose to be administered in the present invention can vary depending on a variety of factors to a great extent, such as target cells, biological types or their tissues, the general condition of the subject to be treated, the route of administration, the mode of administration, etc.

[0037] In another specific embodiment of the present invention, the subjects of drug administration can be humans and non-human mammals, such as mice, rats, guinea pigs, rabbits, dogs, monkeys, gorillas, etc.

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

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

[0040] The "therapeutically effective amount" refers to the amount of active compounds or agents, including the compounds of the present invention, which can cause the biological or medical response of the tissue system, animal or human being sought by the researcher, veterinarian, doctor or other medical personnel, including the alleviation or partial alleviation of the symptoms of the disease, syndrome, condition or disorder being treated. It must be recognized that the optimal dosage and interval of the active ingredients of the present invention are determined by their properties and external conditions such as the form, route and site of administration and the specific mammal being treated, and this optimal dosage can be determined by conventional techniques. It must also be recognized that the optimal course of treatment, that is, the daily dosage of the compound at the same time over a specified period of time, can be determined by methods known in the art.

[0041] The present invention is further described below in conjunction with specific examples, which are only for explaining the present invention and do not limit its content. Any simple modification, equivalent changes and modifications made to the implementation methods based on the technical essence of the present invention are within the scope of the technical solution of the present invention.

[0042] Example

[0043] Materials and methods:

[0044] 1. Establishment of schizophrenia animal model

[0045] C57BL / 6J mice were used, with 10 mice in each group, a total of four groups, including a control group, a schizophrenia group, a risperidone group alone, and an idecalcitol combined with risperidone group. The mice in the control group were intraperitoneally injected with phosphate-buffered saline (PBS) daily and gavaged with glycerol. The other three groups of mice were intraperitoneally injected with dizocilpine (MK-801) for 14 consecutive days at a dose of 1 mg / kg / day, and a schizophrenia model was successfully established. Subsequently, MK-801 was continuously administered in the same manner until the end of the modeling process to maintain the manifestation of mental symptoms. After the schizophrenia model was successfully established, the risperidone monotherapy group was intraperitoneally injected with risperidone at a dose of 1 mg / kg / day for 8 weeks, and glycerol was gavaged at the same time. After verifying that the SZ model was successfully established, the idecalcitol combined with risperidone treatment group was intraperitoneally injected with risperidone at a dose of 1 mg / kg / day, and idecalcitol was gavaged at a dose of 50 ng / kg / day, and the treatment course was 8 weeks.

[0046] 2. Cell Culture

[0047] Hippocampal neuron cell line HT22 cells were cultured in high-glucose DMEM medium containing 10% fetal bovine serum and 1% antibiotics (penicillin, streptomycin). Osteoblast cell line MC3T3-E1 cells were cultured in α-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 experiment

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

[0050] 4. Three-Box Social Experiment

[0051] The test mice were first placed in an empty device (i.e., a three-chamber box consisting of three interconnected liner compartments) and accustomed to the scene. On the test day, the test mice were accustomed to the central compartment with the door closed for 5 minutes. After the habituation phase, the test mice were trained and tested to evaluate social cognitive ability. The training phase allowed the test mice to freely explore the compartment with the same type of mice or another empty compartment for 10 minutes. In the test phase conducted 10 minutes later, the test mice were allowed to contact known mice (same mice as in the training phase) and strange mice in another external compartment to test their interaction preference, and the experimental test lasted for 10 minutes. The close contact time between the test mice and known mice and strange mice was detected and statistically analyzed.

[0052] 5. Water Maze Experiment

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

[0054] 6. Micro-CT

[0055] After modeling, Micro-CT scanning was performed and the femur was removed by sterile dissection. Subsequently, these samples were scanned using a Micro-CT analysis system and three-dimensional images were reconstructed at a resolution of 10 microns.

[0056] 7. HE staining

[0057] HE staining was used to observe histological changes. After dewaxing 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 sealed. Thereafter, the sections were observed and images were obtained under an optical microscope.

[0058] 8. Nissl staining

[0059] After the sections were dewaxed and hydrated, Nissl stain was added to the sections on the slides, coverslips were added, and staining was performed for 10 minutes. After staining, the slides were thoroughly rinsed with distilled water to remove unbound dyes and ensure a clear field of view. The rinsed slides were then sealed with gum and images were obtained under an optical microscope.

[0060] 9. Immunohistochemical staining and TRAP staining

[0061] After the paraffin sections were dewaxed and hydrated, the sections were pretreated with 0.3% hydrogen peroxide for 30 minutes and then blocked with 1% bovine serum albumin (BSA) / PBS for 30 minutes at room temperature. The sections were then incubated with the corresponding antibodies overnight. The next day, the sections were rinsed in PBS after rewarming, and then the sections were stained with secondary antibodies for 1 hour at room temperature. DAB colorimetric solution was then prepared, added dropwise to the sections, and observed under an optical microscope and stopped in time. The sections stained with the ALP&TRAP double staining method were rinsed in double distilled water after color development, and then the TRAP staining solution was added dropwise and reacted at room temperature for 15 minutes until red staining was observed. Finally, all 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 homogenate, take the tissue and weigh its mass. Add 1 mL of 0.9% saline for every 200 mg, grind thoroughly and transfer the tissue homogenate to an EP tube. Centrifuge the homogenate at 12,000 rpm for 10 min at 4°C. Take the supernatant for ELISA. Use relevant kits to detect the levels of prolactin, norepinephrine, and γ-aminobutyric acid in the samples, and use an enzyme reader to evaluate the ELISA results.

[0064] 11. Transmission Electron Microscopy

[0065] The ultrastructure of HT22 cells and MC3T3-E1 cells was observed by transmission electron microscopy. The 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. The tissue was cut into thin sections using an ultramicrotome (EMUC7, Leica). Ultrathin sections (60-80 nm) placed 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 in confocal dishes 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, and then the cells were imaged on a confocal microscope.

[0068] 13.BODIPY 581 / 591C11

[0069] To assess lipid-ROS levels, cells were seeded into confocal microplates and stained with 5 μM of the lipid peroxidation sensor BODIPY 581 / 591C11 for 30 min at 37°C, followed by replacement with PBS and observation on a confocal microscope.

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

[0071] The cultured cells were fixed with 4% paraformaldehyde for 15 min and stained with alkaline phosphatase to detect the alkaline phosphatase activity of osteoblasts. The cultured cells were fixed with 4% paraformaldehyde for 15 min and then stained with 1% AR staining solution to detect the degree of osteoblast mineralization. Imaging was obtained by optical microscopy (CKX-41, Olympus Corporation, Japan).

[0072] result:

[0073] 1. Idecalcitol restores γ-aminobutyric acid levels by improving risperidone-induced ferroptosis of hippocampal neurons, 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 ( Figure 1A). Compared with the control group, the total moving distance and average speed of mice in the MK-801 group increased significantly, and the total stagnation time decreased significantly. The risperidone group showed improvement, and the combination with idecalciferol further reversed the hyperactive behavior ( Figure 1 B) These results suggest that idecalcitol synergizes with risperidone to improve MK-801-induced hyperactive behavior.

[0075] We used a three-box social experiment to evaluate the effects of idecalcitol combined with risperidone on the social cognitive ability of mice to explore its efficacy on the negative symptoms of schizophrenia ( Figure 1 C). In the social preference test, the exploration time of the MK-801 group in the chamber containing the new mouse was significantly reduced compared with the control group, while there was no significant difference between the risperidone group and the MK-801 group. Idecalciferol combined with risperidone can significantly alleviate the social avoidance induced by MK-801 ( Figure 1 D) These results indicate that idecalcitol combined with risperidone effectively reverses MK-801-induced negative symptoms.

[0076] During the training of the water maze test, the latency of the MK-801 group mice to find the platform was longer than that of the control group, while the risperidone group could not improve the latency of the MK-801 group. However, the combination of idecalciferol and risperidone shortened the latency of the schizophrenia mice to find the platform ( Figure 1 EF). In addition, during the test, compared with the MK-801 group, there was no significant difference in the number of platform crossings and platform stay time in the risperidone group, while the idecalciferol group improved the cognitive ability of schizophrenia mice ( Figure 1 G).

[0077] The hippocampus is an important brain region for regulating schizophrenia. GABA neurons in the hippocampus regulate the balance of neurotransmitters in the brain by secreting GABA, thereby affecting the occurrence and development of various psychiatric symptoms. In our study, Nissl staining was used to evaluate the activity and function of hippocampal neurons. The activity of hippocampal neurons in the MK-801+risperidone group was decreased compared with the use of MK-801 alone, while idecalciferol partially rescued this damage ( Figure 1 H).

[0078] The effect of hippocampal neuron damage on the instability of neurotransmitters in the brain was further tested by ELISA. The levels of neurotransmitters in mouse brain tissue homogenates were detected. The results showed that compared with the MK-801 group, the level of inhibitory neurotransmitter γ-aminobutyric acid in the hippocampus of the risperidone group was lower, while the combined idecalciferol group alleviated the loss of γ-aminobutyric acid content in the hippocampus ( Figure 1 I).

[0079] In order to explore the specific mechanism of the decline of γ-aminobutyric acid in the hippocampus, we selected the hippocampal neuronal cell line HT22 for in vitro experiments. Studies have reported that this cell line can synthesize and secrete γ-aminobutyric acid, which can meet our research objectives. In order to explore the reasons for the decrease in cell activity under the action of risperidone, transmission electron microscopy was used to observe the ultrastructure of each group. The results showed that after the administration of risperidone, compared with the group given MK-801 alone, the mitochondrial length of HT22 cells was shortened, mitochondrial vacuolation was accompanied by increased mitochondrial electron density, showing typical characteristics of ferroptosis, and pretreatment with idecalciferol significantly alleviated the poor ultrastructure of cell mitochondria ( Figure 1 J).

[0080] To verify the functional role of risperidone-induced hippocampal neuronal ferroptosis and idecalcitol in hippocampal neuronal injury, we used the mitochondrial probe Mito-Tracker and the mitochondrial ferrous ion probe Mito-FerroGreen to co-stain HT22 cells, and further used BODIPY 581 / 591C11 to detect lipid peroxidation levels. The results showed that compared with the MK-801 group, risperidone significantly aggravated iron accumulation in mitochondria and cellular lipid peroxidation in HT22 cells, and these ferroptosis characteristics could be alleviated by idecalcitol pretreatment ( Figure 1 KL).

[0081] 2. Idecalcitol improves osteoporosis in risperidone-treated schizophrenia mice via a direct pathway involving neural pathways and inhibition of osteoblast ferroptosis

[0082] In addition to psychiatric symptoms, we further investigated the effects of risperidone and idecalcitol on bone remodeling in MK-801-induced schizophrenia mice. Micro-CT showed that MK-801-induced schizophrenia mice had reduced bone mass compared with the CON group, and the bone mass of schizophrenia mice treated with risperidone was further reduced, while the bone mass of mice pretreated with idecalcitol was significantly restored. Compared with the risperidone group, the number and thickness of trabeculae in the idecalcitol group were increased ( Figure 2 A), and further HE and Masson staining obtained similar results ( Figure 2 B). In order to clarify the bone remodeling regulatory mechanism of idecalcitol in restoring bone mass, we used immunohistochemical staining to detect bone formation and bone resorption-related factors. As expected, the immunohistochemical results showed that risperidone administration reduced the expression levels of osteogenic-related factors RUNX2 and ALP in schizophrenia mice, while the combination with idecalcitol significantly promoted bone formation in MK-801+risperidone group mice. In addition, risperidone promoted bone resorption in schizophrenia mice, while idecalcitol inhibited the overactivation of bone resorption levels in mice ( Figure 2C). Therefore, prophylactic administration of idecalcitol can significantly increase bone formation in the MK-801+risperidone group of mice and partially restore bone mass.

[0083] In order to clarify the mechanism of osteoporosis in risperidone-treated schizophrenia mice and the regulatory effect of idecalcitol on it, we explored the neural pathway and direct effect. The first is the effect of neural pathway on bone metabolism. Studies have shown that γ-aminobutyric acid levels have an important regulatory effect on bone formation. Considering the differences in γ-aminobutyric acid content in the hippocampus between the groups ( Figure 1 I), therefore, idecalcitol administration may promote the osteogenic function of MK-801+risperidone group mice by improving central γ-aminobutyric acid. In addition, studies have shown that risperidone can regulate neural circuits by antagonizing DA receptors and 5-HT receptors, thereby inducing hyperprolactinemia and sympathetic nerve excitement, thereby inducing bone metabolic homeostasis. We used ELISA to detect the levels of prolactin and norepinephrine in femoral tissue homogenate, and found that idecalcitol downregulated the high plasma prolactin levels and high norepinephrine levels induced by risperidone ( Figure 2 DE).

[0084] In addition, to explore the direct effect of edecalcitol on bone formation in risperidone-treated schizophrenia mice, the osteoblast MC3T3-E1 cell line was selected 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. The results showed that ED-71 pretreatment partially alleviated risperidone-induced osteogenic dysfunction and increased the formation of mineralized nodules ( Figure 2 F). In order to clarify the mechanism of drug effects on cell function, transmission electron microscopy was used to observe the ultrastructure of each group. The results showed that after risperidone administration, compared with the group given MK-801 alone, the length of osteoblast mitochondria was shortened, mitochondrial vacuolization was accompanied by increased mitochondrial electron density, showing typical characteristics of ferroptosis, while idecalciferol pretreatment significantly alleviated the poor ultrastructure of cell mitochondria ( Figure 2 G).

[0085] To verify the functional role of risperidone-induced osteoblast ferroptosis and idecalcitol in osteoblast injury, we used the mitochondrial probe Mito-Tracker and the mitochondrial ferrous ion probe Mito-FerroGreen to co-stain MC3T3-E1 cells, and further used BODIPY 581 / 591C11 to detect lipid peroxidation levels. The results showed that compared with the MK-801 group, risperidone significantly aggravated iron accumulation in mitochondria and cellular lipid peroxidation in MC3T3-E1 cells, and this ferroptosis feature could be alleviated by idecalcitol pretreatment ( Figure 2 HI).

[0086] In summary, idecalcitol can restore the homeostasis of neurotransmitters in the brain by inhibiting the ferroptosis of γ-aminobutyric acid neurons in the hippocampus, thereby synergizing with risperidone to improve schizophrenia; at the same time, idecalcitol can alleviate risperidone-induced osteoporosis by regulating neural pathways and preventing the direct pathway of ferroptosis of osteoblasts. Therefore, this study is expected to bring new breakthroughs in the treatment of schizophrenia, and achieve the clinical efficacy of "killing two birds with one stone" by developing the combination of idecalcitol and risperidone, in order to provide new hope and direction for the treatment of schizophrenia patients.

[0087] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. The use of idecalcitol in the preparation of a drug for promoting the therapeutic effect of risperidone on schizophrenia while preventing and treating the side effects of risperidone.

2. The use according to claim 1, characterized in that: The promoting effect of risperidone on the treatment of schizophrenia is specifically manifested in that the γ-aminobutyric acid level is restored by improving the ferroptosis of γ-aminobutyric acid neurons in the hippocampus induced by risperidone, thereby cooperating with risperidone to alleviate the symptoms of schizophrenia.

3. The use according to claim 1, characterized in that: The side effects of risperidone include bone loss mediated by risperidone, which in turn causes osteoporosis; Furthermore, the idecalcitol prevents and treats the side effects of risperidone in the following specific manifestations: the idecalcitol alleviates risperidone-induced osteoporosis by directly regulating neural pathways and preventing osteoblast ferroptosis.

4. Use of idecalcitol combined with risperidone in the preparation of drugs for the treatment of schizophrenia.

5. The use according to claim 4, characterized in that: When the idecalcitol and risperidone are used together, the mass ratio of the two is 1-100×10 -6 :

1.

6. The use according to claim 4, characterized in that: The treatment of schizophrenia is specifically manifested as follows: while idecalcitol is combined with risperidone to synergistically treat schizophrenia, idecalcitol is used to prevent and treat the side effects mediated by risperidone.

7. The use according to claim 6, characterized in that The synergistic treatment of schizophrenia by idecalcitol and risperidone is specifically manifested in that idecalcitol restores the level of γ-aminobutyric acid by improving risperidone-induced ferroptosis of hippocampal neurons, thereby synergizing with risperidone to alleviate the symptoms of schizophrenia.

8. The use according to claim 6, characterized in that The side effects of risperidone include bone loss mediated by risperidone, which in turn causes osteoporosis; Furthermore, the idecalcitol prevents and treats the side effects of risperidone in the following specific manifestations: the idecalcitol alleviates risperidone-induced osteoporosis by directly regulating neural pathways and preventing osteoblast ferroptosis.

9. A pharmaceutical composition for treating schizophrenia, characterized in that: The active ingredients of the pharmaceutical composition at least include idecalcitol and risperidone; when the idecalcitol and risperidone are used in combination, the mass ratio of the two is 1-100×10 -6 :

1.

10. The pharmaceutical composition according to claim 9, characterized in that The pharmaceutical composition further comprises at least one non-pharmaceutically active ingredient.

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