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 be necessarily regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those 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 5-hydroxytryptamine (5-HT) system and the dopamine (DA) system and have been shown to partially relieve the positive and negative symptoms of schizophrenia, but have weak efficacy in cognitive dysfunction. Although risperidone can partially block the binding of transmitters by antagonizing transmitter receptors, it cannot fundamentally restore the balance of transmitters in the brain, and high-risk doses have certain neurotoxicity, so its efficacy has the limitation of treating the symptoms but not the root cause.

[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 interneurons of schizophrenia patients receiving high-risk risperidone treatment may be damaged, leading to an aggravation of the disorder of neurotransmitter levels in the brain.

[0005] In addition, the use of antipsychotic drugs can produce other side effects, including osteoporosis, decreased bone density and increased risk of fractures. Studies have shown that risperidone causes 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 and overactivates the sympathetic nervous system to indirectly induce osteoporosis. Studies have reported that the type of osteoporosis induced by risperidone is mainly low-conversion type, and high-conversion osteoporosis only occurs when long-term hyperprolactinemia feedback induces hypogonadism. In addition, the level of γ-aminobutyric acid, which is closely related to mental symptoms, has a regulatory effect on osteogenic function. When the γ-aminobutyric acid content is deficient, it will lead to bone formation disorders, 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 pathways and direct pathways of risperidone-induced bone loss deserve further exploration and understanding.

[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 excessive induction of autophagy. Many pathological processes involved in ferroptosis, such as abnormal fatty acid and iron metabolism and ROS accumulation, depend 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 amphiphilic weakly basic cationic drugs of antipsychotic drugs. This small molecule with hydrophobic weak base properties is called a lysosomal drug, which accumulates significantly in lysosomes through a mechanism called ion capture. The 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 1 The present invention shows that idecalcitol in the embodiment of the present invention improves risperidone-induced hippocampal neuronal ferroptosis and restores γ-aminobutyric acid levels, thereby cooperating with risperidone to relieve schizophrenia symptoms. (A) Open field test route map and heat map. (B) Statistical analysis of total moving distance, total stagnation time and average moving speed. (C) Three-box social test route map and heat map. (D) Statistical analysis of social ability in the first stage and social novelty in the second stage. (E) Water maze test route map. (F) Statistical analysis of the number of platform crossings and platform residence time during the incubation period and the fifth day test. The above data are expressed as mean ± standard deviation (n = 6). (G) HE staining and Nissl staining of the CA1 region of the hippocampus. (H) Statistical analysis of γ-aminobutyric acid levels in mouse hippocampal tissue homogenates; statistical analysis of DA levels in mouse prefrontal cortex tissue homogenates; statistical analysis of 5-HT levels in mouse hippocampal ventral subiculum tissue homogenates. Data are expressed as mean ± standard deviation (n = 3). (I) Transmission electron microscopy image of mouse hippocampal neuronal cell line HT22 cells. (J) Fluorescence image of HT22 cells co-stained with mitochondrial probe and ferrous ion probe. (K) Fluorescence image of HT22 cells with lipid peroxidation probe;

[0020] Figure 2The 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 MMP9 immunohistochemical staining images. (D) Statistical analysis of mouse plasma γ-aminobutyric acid levels. (E) Statistical analysis of mouse plasma prolactin levels. (F) Statistical analysis of mouse plasma norepinephrine. The above data are expressed as mean ± standard deviation (n = 3). (G) Transmission electron microscopy image of MC3T3-E1 cells. (H) ALP and Alizarin red staining images of MC3T3-E1 cells. (I) Fluorescence image of MC3T3-E1 cell mitochondrial probe co-stained with ferrous ion probe. (J) Fluorescence image 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, including a control group, a schizophrenia group, a risperidone group, and an idecalciferol combined with risperidone group. The control group was intraperitoneally injected with the corresponding volume of PBS and gavaged with the corresponding volume of triglycerides every day. The other three groups were intraperitoneally injected with 1 mg / kg / day of dizocilpine (MK-801) for 35 days to construct a schizophrenia mouse model in a drug-induced manner. The risperidone group was given an intraperitoneal injection of 1 mg / kg / day of risperidone 7 days after MK-801 administration for 28 days. The idecalciferol combined with risperidone group was given an intraperitoneal injection of 1 mg / kg / day of risperidone 7 days after MK-801 administration, and idecalciferol was gavaged at 50 ng / kg / day for 28 days.

[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). Preosteoblast 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 of 5% CO2. At 80% confluence, the cells were digested with trypsin-EDTA and passaged.

[0048] 3. Open field experiment

[0049] The open field test apparatus consisted of a box with dimensions of 40×40×40 cm (length×width×height). The apparatus was made of black high-density polyethylene plates that were fastened together and placed on a plastic base. The behavior of the mice was recorded with a camera mounted above the open field. The heat map was generated using an animal motion trajectory tracking heat map generator. The total movement distance, total stagnation time, and average movement speed of the test mice were 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. The test was 10 minutes / day for a period of 3 days. 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 tested to evaluate social ability and social novelty after 10 minutes. The first phase of social ability detection was to test the social or exploratory preference of the test mice to the compartment with the same type of mice or another empty compartment, and the test lasted for 10 minutes. The second phase social novelty detection carried out after 10 minutes was to contact the test mice with known mice (same mice in the social ability detection phase) and the strange mice in another external compartment, test their interaction preference, and the experimental test lasted for 10 minutes.

[0052] 5. Water Maze Experiment

[0053] Animals were tested in a circular tank with a diameter of 1.2 m located in a maze room. The platform (10 cm in diameter) 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 4-day learning phase and a fifth-day testing phase. During the learning phase, the animals were placed in a 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 gently pushed forward from the base of its tail and guided to the target position, allowing the mouse to explore the maze clues on the platform for 10 seconds. In the testing phase, the hidden platform was removed, the mouse was allowed to explore freely for 60 seconds, video recorded, and the number of cross-platforms and the 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 plasma samples, collect whole blood into a test tube containing an anticoagulant, mix well, place on ice, centrifuge at about 1500×g at 4°C for 10 minutes, and take the yellow or light yellow supernatant to obtain plasma. For brain tissue homogenate, take the hippocampus, prefrontal cortex, and ventral subiculum of the hippocampus and weigh their masses. Add 1mL of 0.9% saline to every 200mg of brain tissue homogenate, grind thoroughly, transfer the tissue homogenate to an EP tube, and centrifuge the homogenate at 12000rpm at 4°C for 10min. Take the supernatant for ELISA detection. Use relevant kits to detect the levels of prolactin, norepinephrine, GABA, DA, and 5-HT in the samples, and use an enzyme reader to evaluate the ELISA results at a wavelength of 450nm.

[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. Ferrous ion detection

[0067] Ferro Orange is a special cell dye that does not emit fluorescence when unbound, but emits fluorescence when combined with ferrous ions (Fe 2+ ) shows a fluorescent signal when it binds. 2+The cells were inoculated in a confocal dish and incubated with iron orange (1 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 TM 581 / 591C11

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

[0070] 14. Alkaline phosphatase (ALP) staining and Alizarin red (AR) 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 1 A). 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 sociability and social novelty of mice to explore its efficacy on the negative symptoms of schizophrenia ( Figure 1C). 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, risperidone, and mice treated with idecalciferol combined with risperidone. 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 5-day training of the water maze test, the latency of the mice in the MK-801 group to find the platform was longer than that in the control group, while the latency of the MK-801+risperidone group was shorter than that of the MK-801 group. On this basis, idecalciferol further shortened the latency ( Figure 1 E). In addition, in the test on the fifth day, compared with the MK-801 group, the number of crossing the platform and the duration of staying on the platform increased in the risperidone group, and the idecalciferol group further improved the cognitive ability of mice ( Figure 1 F).

[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 of hippocampal neurons. The survival rate of hippocampal neurons in the MK-801+risperidone group was lower than that in the mice treated with MK-801 alone ( Figure 1 D), while idecalcitol partially rescued this damage ( Figure 1 G).

[0078] To further examine the effect of hippocampal neuron damage on the instability of neurotransmitters in the brain, the ELISA method was used to detect the levels of multiple neurotransmitters in mouse brain tissue homogenates. The results showed that compared with the MK-801 group, the inhibitory neurotransmitter γ-aminobutyric acid level was higher in the hippocampus of the risperidone group, while the combined idecalcitol group alleviated the loss of γ-aminobutyric acid content in the hippocampus. In contrast, schizophrenia-related neurotransmitters including DA in the prefrontal cortex and 5-HT in the ventral subiculum of the hippocampus increased after risperidone administration, while idecalcitol pretreatment alleviated their excessive increase ( Figure 1 H).

[0079] In order to explore the specific mechanism of the decrease of γ-aminobutyric acid in the hippocampus and the subsequent excessive levels of DA and 5-HT, 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 length of mitochondria in HT22 cells was shortened, the mitochondrial membrane was ruptured, and the membrane density was increased. The cristae were reduced or even disappeared, showing the typical characteristics of ferroptosis, and pretreatment with idecalciferol significantly alleviated the adverse ultrastructure of cell mitochondria ( Figure 1 I).

[0080] To verify the functional role of risperidone-induced ferroptosis in hippocampal neurons and idecalcitol in hippocampal neuronal injury, we used the mitochondrial probe Mito-Tracker and the ferrous ion probe FerroOrange to co-stain HT22 cells, and further used BODIPY TM 581 / 591C11 detected 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 this ferroptosis feature could be alleviated by pretreatment with idecalciferol ( Figure 1 JK).

[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 significantly increased ( Figure 2 A), and further HE and Masson staining obtained similar results ( Figure 2B). 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 the use of idecalcitol significantly promoted bone formation in the MK-801+risperidone group of mice. Specifically, idecalcitol increased the positive expression of RUNX2 and ALP immunohistochemical staining in schizophrenia mice administered with risperidone. Interestingly, risperidone inhibited bone resorption in schizophrenia mice, while idecalcitol partially restored the bone resorption level in mice ( Figure 2 C). In conclusion, these results indicate that prophylactic administration of idecalcitol can significantly increase bone formation and partially restore bone mass in mice in the MK-801+risperidone group.

[0083] In order to clarify the mechanism of osteoporosis in schizophrenia mice treated with risperidone and the regulatory effect of idecalcitol on it, we explored the neural pathways and direct effects. The first is the effect of neural pathways on bone metabolism. Studies have shown that γ-aminobutyric acid levels have an important regulatory effect on bone formation. Considering that there are differences in γ-aminobutyric acid content in the brain between groups, we further used ELISA to detect the plasma γ-aminobutyric acid level of each group. The results showed that the γ-aminobutyric acid content in plasma showed a trend similar to that in the brain, and idecalcitol administration alleviated the low γ-aminobutyric acid level in the bones of schizophrenia mice treated with risperidone ( Figure 2 D). 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 excitation, thereby inducing bone metabolic homeostasis. We used ELISA to detect the levels of prolactin and norepinephrine in plasma, and the results showed that idecalcitol downregulated the high plasma prolactin levels and high norepinephrine levels induced by risperidone ( Figure 2 EF).

[0084] In addition, the direct effect of idecalcitol on bone formation in schizophrenia mice treated with risperidone was investigated, and the preosteoblast MC3T3-E1 cell line was used for in vitro experiments. The ultrastructure of each group was observed using a transmission electron microscope. The results showed that after risperidone administration, compared with the group given MK-801 alone, the length of osteoblast mitochondria was shortened, the mitochondrial membrane was ruptured, and the membrane density was increased. The cristae were reduced or even disappeared, showing typical characteristics of ferroptosis. Pretreatment with idecalcitol significantly alleviated the adverse ultrastructure of cell mitochondria ( Figure 2 G). In order to clarify whether drug damage to cell ultrastructure would affect cell function, 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 H).

[0085] To verify the functional role of risperidone-induced osteoblast ferroptosis and idecalcitol in osteoblast injury, we co-stained HT22 cells with the mitochondrial probe Mito-Tracker and the ferrous ion probe FerroOrange, and further used BODIPY TM 581 / 591C11 detected 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 pretreatment with idecalciferol ( Figure 2 IJ).

[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: restoring the level of gamma-aminobutyric acid by improving risperidone-induced ferroptosis of hippocampal neurons, thereby cooperating with risperidone to alleviate schizophrenia symptoms.

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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