Application of oryzanol combined with ED-71 in prevention and treatment of angiogenesis disorder in early clinical stage of Alzheimer disease

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of oryzanol combined with ED-71 in prevention and treatment of angiogenesis disorder in the early clinical stage of Alzheimer's disease. Specifically, the invention discovers and verifies for the first time through research that in the early clinical stage of AD, sympathetic nerve hyperexcitation may cause H-type vascular endothelial cell senescence and hinder vascular osteogenesis by interfering with a PKM2-mediated glycolysis process, and finally causes bone mass loss. Oryzanol and ED-71 can synergistically improve vascularized osteogenesis damage of the Alzheimer's disease, so that the pharmaceutical composition is used for effectively preventing and treating angiogenesis disorder of the Alzheimer's disease in the early clinical stage, and has good practical application value.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to the application of oryzanol combined with ED-71 in preventing and treating vascular osteogenesis disorder in the preclinical stage of Alzheimer's disease. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance 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 those skilled in the art.

[0003] As the population ages, the incidence of Alzheimer's disease (AD) continues to rise. According to the "China Alzheimer's Disease Report 2024," AD and other dementias are now the fifth leading cause of death among urban and rural residents in China.

[0004] Clinical epidemiological data show that the incidence of fractures in AD patients is 2.5 times higher than that of their peers with normal intelligence. AD patients themselves suffer tremendous pain due to the disease, and the occurrence of fractures is a serious threat to their life and health, becoming a key factor in the deterioration of the disease and even death. However, the pathological connection between fractures and AD is still unclear. AD has a long incubation period, called the preclinical stage of AD. Therapeutic intervention during this period before clinical cognitive impairment appears is a golden opportunity to slow the progression of the disease. Therefore, if we can focus on the deep connection between H-type vascular damage and bone loss in the preclinical stage of AD, it is expected to open up new paths for the prevention and / or treatment of AD and fractures and provide effective intervention strategies. Summary of the Invention

[0005] In response to the deficiencies in the above-mentioned prior art, the inventors, after long-term technical and practical exploration, provide the use of oryzanol combined with ED-71 in preventing and treating vascular osteogenesis disorders in the preclinical stage of Alzheimer's disease. Specifically, the present invention has discovered and confirmed for the first time through research that in the preclinical stage of AD, sympathetic nerve hyperexcitation may interfere with the PKM2-mediated glycolysis process, leading to the aging of H-type vascular endothelial cells and hindering vascular osteogenesis, ultimately causing bone loss. Oryzanol and ED-71 can synergistically improve the damage to vascularized osteogenesis in Alzheimer's disease, thereby being used for the effective prevention and treatment of vascular osteogenesis disorders in the preclinical stage of Alzheimer's disease. Based on the above research results, the present invention is completed.

[0006] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0007] The first aspect of the present invention provides the use of oryzanol combined with ED-71 in the preparation of a drug for preventing and / or treating vascular osteogenesis disorder in the preclinical stage of Alzheimer's disease.

[0008] Specifically, the prevention and / or treatment of vascular osteogenesis disorder in the preclinical stage of Alzheimer's disease is specifically manifested by any one or more of the following (a)-(f):

[0009] (a) Reduce the expression level of norepinephrine;

[0010] (b) inhibiting sympathetic nerve overactivation-induced H-type endothelial cell senescence;

[0011] (c) Increase Osterix + osteoblast progenitor cell number;

[0012] (d) enhance mineralized bone formation;

[0013] (e) Improve bone density and structure;

[0014] (f) Improve the impairment of vascularized bone formation in the preclinical stage of Alzheimer's disease.

[0015] The second aspect of the present invention provides a pharmaceutical composition for preventing and / or treating vascular osteogenesis disorder in the preclinical stage of Alzheimer's disease, wherein the active ingredients of the pharmaceutical composition include at least oryzanol and ED-71.

[0016] The third aspect of the present invention provides a method for preventing and / or treating vascular osteogenesis disorder in the preclinical stage of Alzheimer's disease, comprising administering the above-mentioned pharmaceutical composition to a test animal.

[0017] Compared with the existing technical solutions, the above one or more technical solutions have the following beneficial effects:

[0018] The above technical solution was proposed for the first time and confirmed by research. It shows that in the early clinical stage of AD, excessive sympathetic nerve excitation may interfere with the PKM2-mediated glycolysis process, leading to the aging of type H vascular endothelial cells and hindering vascular osteogenesis, ultimately causing bone loss and thus forming a vascular osteogenesis disorder. It was then proposed to use oryzanol and ED-71 in combination to explore the development of effective prevention and treatment strategies for bone loss in the early clinical stage of AD. Therefore, it has important clinical significance and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The accompanying drawings, 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.

[0020] Figure 1 In the present embodiment, the H vascular damage in the early stage of AD clinical stage is correlated with sympathetic nerve hyperexcitation, wherein a. 3-month-old and 4-month-old APP / PS1 mice and wild-type mice EMCN hi CD31hi Confocal images of cells and quantitative histomorphometric analysis of H-type vessel area. b. Detection of EMCN in bone by flow cytometry + CD31 + Cell ratio. c. Confocal image of Aβ in bone vessels. d. ELISA detection of Aβ in serum and bone tissue. e. ELISA detection of norepinephrine (NE) in serum and bone tissue. f. Confocal image of CD31 co-stained with tyrosine hydroxylase (TH).

[0021] Figure 2 In the present invention, inhibiting sympathetic nerve overactivation can reverse the damage of vascular bone formation in preclinical Alzheimer's disease mice. Propranolol and 6-hydroxydopamine (6-OHDA) were used to inhibit sympathetic nerve overactivation in APP / PS1 mice. hi CD31 hi Confocal images. b. CD31 hi Perivascular Osterix + (Green) Confocal images of osteoprogenitor cell numbers. c. Immunohistochemical (IHC) analysis of alkaline phosphatase (ALP) and RUNX2 in the tibia. d. Assessment of bone mass by μCT. e. Schematic diagram of magnetic bead sorting of osteoendothelial cells from the femoral and tibial metaphyseal regions using an EMCN antibody. f. ELISA assays for Noggin and TGF-β. g. Images and statistical analysis of alkaline phosphatase (ALP) and Alizarin Red (ARS) staining.

[0022] Figure 3 In the embodiment of the present invention, excessive sympathetic nerve activation impairs vascular bone formation in the preclinical stage of Alzheimer's disease mice by promoting H-type endothelial cell aging, wherein: a. Upregulated and downregulated genes in the transcriptome. b. KEGG pathway analysis of vascular endothelial cells treated with control group and NE. c. qRT-PCR detection of P53, P21 and P16 in the control group, 10μM NE, 100μM NE and H2O2 group (cell aging positive control). d. Western blot analysis of P53 and P21. e. SA-β-galactosidase (SA-β-Gal) activity detection and cell immunofluorescence staining of HMGB1. f. β-Gal + Quantitative analysis of endothelial cell number and fluorescence intensity. g. CD31 in APP / PS1 mice + β-Gal +Confocal images of cells. h. qRT-PCR detection of P53, P21, and P16 in bone tissue samples. i. Heat map analysis of inflammatory factor expression. j. Schematic diagram of MiDAS signature. k. Detection of NAD+ / NADH in the control group, NE-treated group, and NE+ICI118551 group. lp Vascular endothelial cells were pretreated with nicotinamide mononucleotide (NMN) and then treated with NE. l. SA-β-Gal activity detection and cell immunofluorescence staining of HMGB1. mn. Cell cycle detection. o. ELISA detection of Noggin and TGF-β. p. ALP staining and ARS staining.

[0023] Figure 4 In the examples of the present invention, PKM2-mediated glycolysis is the key to sympathetic nerve overactivation-induced senescence in H-type endothelial cells, including: a. KEGG pathway classification of control and NE-treated vascular endothelial cells. b. Evaluation of the effect of NE on glucose uptake capacity using a 2-NBDG kit. c. Volcano plot analysis of PKM2 expression after NE treatment. d. Schematic diagram of the glycolysis process. e. Immunofluorescence staining of PKM2 in vascular endothelial cells. f. PKM2 activity assay. g. Pyruvate, lactate, and ATP content assay. hk. Restoration of NE-induced glycolysis homeostasis imbalance using the selective PKM2 activator TEPP-46 and pyruvate supplementation. h. SA-β-Gal activity assay and HMGB1 immunofluorescence staining. i. Lactate and ATP content assay. j. ELISA assay for Noggin and TGF-β. k. ALP staining and ARS staining. ln. Using adeno-associated virus (AAV) encoding PKM2 and pyruvate supplementation to regulate the glycolytic pathway in bone vascular endothelial cells of APP / PS1 mice. l. CD31 in APP / PS1 mice + β-Gal + Confocal images of cells. m. HE staining and Micro-CT imaging. n. Quantitative analysis of bone mass.

[0024] Figure 5The present invention shows that c-Maf participates in the glycolysis process mediated by PKM2 in NE-treated vascular endothelial cells, including: a. Heat map analysis of the top 20 upregulated and downregulated genes in control and NE-treated vascular endothelial cells. b. qRT-PCR detection of c-Maf expression. d. Western blot analysis of cc-Maf expression. d. Cell immunofluorescence staining of c-Maf. Quantitative analysis of ec-Maf fluorescence intensity. f. Prediction of binding targets of c-MAF and PKM2 using the NCBI and JASPAR databases. gn. Verification of the relationship between c-Maf and PKM2 using Nivalenol (a c-Maf-specific inhibitor) and a c-Maf overexpression plasmid. gh. qRT-PCR detection of c-Maf and PKM2. ic-Maf and PKM2 linearity analysis. j. Cell immunofluorescence staining of PKM2. k. Detection of PKM2 activity and pyruvate content. l. Detection of the NAD+ / NADH ratio.

[0025] m. SA-β-Gal activity assay. n. ELISA assay for Noggin and TGF-β in vascular endothelial cells. o. Western blot analysis of ALP, RUNX2, and OCN in osteoblast progenitor cells.

[0026] Figure 6 The present invention shows that oryzanol and ED-71 synergistically improve the damage of vascular bone formation in preclinical Alzheimer's disease mice, including: a. ELISA detection of NE in serum and bone tissue of APP / PS1 mice treated with oryzanol and ED-71. b. Confocal imaging of Aβ in bone vessels. c. Quantitative analysis of Aβ in bone vessels. d. CD31 in APP / PS1 mice. + β-Gal + Confocal images of cells. e.CD31 in APP / PS1 mice + β-Gal + Quantitative analysis of cell number. fg.CD31 hi Perivascular Osterix + (Green) Confocal images and quantitative analysis of osteoblast progenitor cells. hi. Assessment of bone formation using calcein labeling. jl. HE staining, Micro-CT imaging, and quantitative analysis of bone mass. DETAILED DESCRIPTION

[0027] 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.

[0028] 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.

[0029] In a typical embodiment of the present invention, there is provided the use of oryzanol combined with ED-71 in the preparation of a drug for preventing and / or treating vascular osteogenesis disorder in the preclinical stage of Alzheimer's disease.

[0030] Specifically, sympathetic nerve overexcitation may be a key pathway for regulating bone loss in the early clinical stage of AD. Therefore, regulating sympathetic nerve activity is expected to become an important breakthrough in delaying bone loss and preventing AD-related bone lesions. Oryzanol is a mixture of ferulic acid esters mainly composed of triterpenoids (ene) alcohols, which is present in rice oil (rice bran oil). Due to its lipophilicity, oryzanol can pass through the blood-brain barrier intact and act on the autonomic nervous system and endocrine center of the diencephalon, so that the autonomic nervous function is regulated and the balance between the sympathetic and parasympathetic nerves is restored. In addition, oryzanol can regulate the release of sympathetic neurotransmitters and improve the symptoms of autonomic nervous system disorders. Therefore, the applicant considered trying to use oryzanol to improve the symptoms of sympathetic nerve overexcitation in AD mice in the early clinical stage to achieve the effect of preventing bone loss. At the same time, the applicant noted that aging of the H-type vascular endothelium may be the pathological basis for vascular osteogenesis disorders. Based on this, starting from the perspective of inhibiting cell aging, exploring suitable combination drugs, and promoting a "step-by-step synergistic, cause-targeted" treatment strategy is a new way of thinking to solve this problem. Eldecalcitol (ED-71) not only exhibits excellent anti-aging effects. It also has a good effect on various types of osteoporosis, such as diabetic osteoporosis, glucocorticoid osteoporosis, etc. Based on the above considerations, as a bold attempt at early clinical intervention treatment for AD, the applicant combined oryzanol and ED-71 to improve the symptoms of autonomic nervous system disorders through oryzanol and improve the aging phenotype of H-type vascular endothelial cells with ED-71. This two-pronged approach effectively improves vascular osteogenesis disorders to prevent and / or treat AD-related bone loss and fractures, ultimately improving the quality of life of AD patients.

[0031] When the oryzanol and ED-71 are used together, the mass ratio of the two is 0.5-5×10 6 :1; preferably 2×10 6 :1.

[0032] Specifically, the prevention and / or treatment of vascular osteogenesis disorder in the preclinical stage of Alzheimer's disease is specifically manifested by any one or more of the following (a)-(f):

[0033] (a) Reduce the expression level of norepinephrine;

[0034] (b) inhibiting sympathetic nerve overactivation-induced H-type endothelial cell senescence;

[0035] (c) increase the number of Osterix+ osteoblast progenitor cells;

[0036] (d) enhance mineralized bone formation;

[0037] (e) Improve bone density and structure;

[0038] (f) Improve the impairment of vascularized bone formation in the preclinical stage of Alzheimer's disease.

[0039] In another embodiment of the present invention, a pharmaceutical composition for preventing and / or treating vascular osteogenesis disorder in the preclinical stage of Alzheimer's disease is provided, wherein the active ingredients of the pharmaceutical composition include at least oryzanol and ED-71.

[0040] Specifically, the prevention and / or treatment of vascular osteogenesis disorder in the preclinical stage of Alzheimer's disease is specifically manifested by any one or more of the following (a)-(f):

[0041] (a) Reduce the expression level of norepinephrine;

[0042] (b) inhibiting sympathetic nerve overactivation-induced H-type endothelial cell senescence;

[0043] (c) increase the number of Osterix+ osteoblast progenitor cells;

[0044] (d) enhance mineralized bone formation;

[0045] (e) Improve bone density and structure;

[0046] (f) Improve the impairment of vascularized bone formation in the preclinical stage of Alzheimer's disease.

[0047] When the oryzanol and ED-71 are used together, the mass ratio of the two is 0.5-5×10 6 :1; preferably 2×10 6 :1.

[0048] Furthermore, the pharmaceutical composition further comprises at least one non-pharmaceutical active ingredient.

[0049] The inactive ingredients of the drug can be carriers, excipients, diluents, etc. commonly used in pharmacy. Moreover, according to common methods, the drug can be prepared into oral dosage forms, external preparations, suppositories, and sterile injection solutions in the form of powders, granules, suspensions, emulsions, syrups, sprays, etc.

[0050] 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.

[0051] 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.

[0052] In another embodiment of the present invention, the drug of the present invention can be administered into 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 will be appreciated by those skilled in the art that the actual dosage to be administered in the present invention can vary to a great extent depending on a variety of 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, etc.

[0053] In another 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, orangutans, etc.

[0054] In another embodiment of the present invention, a method for preventing and / or treating vascular osteogenesis disorder in the preclinical stage of Alzheimer's disease is provided, comprising administering the above-mentioned pharmaceutical composition to a subject.

[0055] The subject refers to an animal that has been the object of treatment, observation or experiment, preferably a mammal, most preferably a human.

[0056] The present invention will be further described below with reference to specific examples. The following examples are only for the purpose of explaining the present invention and are not intended to limit its content. Any simple modifications, equivalent changes and modifications made to the embodiments based on the technical essence of the present invention are within the scope of the technical solution of the present invention.

[0057] Example

[0058] Experimental methods

[0059] 1. Assessing the correlation between vascular osteogenesis impairment and sympathetic nervous system activation in the preclinical phase of AD

[0060] 1. Assessing H-type vascular damage in the early stages of AD

[0061] (1) Animal model establishment and material collection:

[0062] Three-month-old male APP / PS1 mice were used as a preclinical AD model, and male negative littermates served as a control group. After anesthesia, blood was collected from the eyeballs, centrifuged at 3000 rpm / min for 10 minutes, and serum was separated and stored at -80°C. After the mice were sacrificed, the jaws and leg bones were isolated. The forelimbs were separated and stored in liquid nitrogen, while the mandibles and tibias were fixed in 4% paraformaldehyde.

[0063] (2) Tissue processing and related testing:

[0064] The isolated forelimbs were ground, and the supernatant was extracted. Aβ and NE levels in the isolated serum and bone tissue supernatants were determined by ELISA. The mandibles and tibiae were fixed, decalcified, dehydrated, embedded, and sectioned to prepare 5 μm paraffin sections and 40 μm frozen sections. Paraffin sections were stained with hematoxylin and eosin and Masson staining to observe morphological changes in the mandibular condyle and tibiae in each group. Frozen sections were immunostained for CD31 and EMCN to assess damage to bone H-type vessels; immunostained for CD31 and Aβ to assess Aβ deposition in H-type vessels within the mandibular condyle and tibiae; and immunostained for CD31 and tyrosine hydroxylase (TH) to assess sympathetic nerve activation within the mandibular condyle and tibiae. The separated mandible and part of the tibia were used for Micro-CT scanning and statistical analysis to evaluate the changes in bone mass in each group. The femur was processed by medullary cavity flushing and digestion to obtain single-cell suspension, and the proportion of vascular endothelial cells was detected by flow cytometry.

[0065] 2. Clarify the relationship between sympathetic nerve hyperactivity and vascular osteogenesis impairment in preclinical AD mice

[0066] (1) Animal model establishment and processing:

[0067] Three-month-old male APP / PS1 mice were used as a preclinical model of AD. The mice were divided into the following groups: littermate-matched male negative mice as a blank control group (WT), an experimental control group (APP / PS1), a propranolol-treated group (APP / PS1+PRO), and a 6-hydroxydopamine-treated group (APP / PS1+6-OHDA). Each group consisted of 10 mice. The propranolol-treated group received an intraperitoneal injection of 20 mg / kg of propranolol (PRO) once daily for 4 weeks; the 6-hydroxydopamine-treated group received 80 mg / kg of 6-hydroxydopamine (6-OHDA) intraperitoneally for the first 3 days of treatment and every 2 weeks thereafter. After 4 weeks, the mice were harvested using the same method as above.

[0068] (2) Tissue processing and related testing:

[0069] Mandibles and tibiae were fixed, decalcified, dehydrated, embedded, and sectioned, and paraffin and frozen sections were prepared. Paraffin sections were stained with hematoxylin and eosin (HE) and masson staining to observe morphological changes in the mandibular condyle and tibiae of each group. Immunohistochemical staining was used to assess osteoblast activity, including ALP and RUNX2. Frozen sections were immunostained for CD31 and EMCN to assess the presence of bone H-type vessels. Immunostained for CD31 and TH to assess sympathetic nerve function in the mandibular condyle and tibiae. Immunostained for CD31 and Osterix to assess changes in the number of osteoprogenitor cells surrounding the H-type vessels in the mandibular condyle and tibiae. Isolated mandibles and a portion of tibiae were used for micro-CT scanning and statistical analysis to assess bone mass changes in each group. Femora were screened for specificity of the bone H-type vessel endothelium.

[0070] (3) Cell extraction and culture:

[0071] a. Extraction and identification of bone H-type vascular endothelial cells: Remove the muscles and periosteum around the femur. Flush the bone marrow cavity, collect the flushing fluid, and cut the diaphysis and epiphysis into pieces. Use collagenase digestion method with Dynabeads TM Sheep anti-rat IgG magnetic beads and EMCN antibodies were used for specific selection and culture of bone H-type endothelial cells. At passage 3, cells were identified by immunofluorescence co-staining with CD31 and ENCM.

[0072] b. Extraction of osteoprogenitor cells: Osteoprogenitor cells were extracted from the leg bones of 4-week-old C57BL / 6 mice.

[0073] (4) Vascular osteogenesis function test:

[0074] Osteogenic induction medium was prepared by culture of magnetically sorted H-type vascular endothelial cells, and the supernatant was mixed with standard osteogenic induction medium at a 1:1 ratio. RNA was extracted from osteoprogenitor cells on day 7 of osteogenic induction, and total protein was extracted on day 14 of osteogenic induction. Expression of osteogenic-related factors such as ALP, RUNX2, and OCN was assessed by qRT-PCR and western blot. ALP staining was performed on day 7 of osteogenic induction and on day 21 of osteogenic induction to assess the effects of sympathetic nerve hyperactivity on vascular osteogenesis.

[0075] 2. Exploring the relationship between H vascular endothelial cell senescence and vascular osteogenesis disorders in the preclinical stage of AD

[0076] 1. Elucidate the pathological basis of H-type vascular damage driven by sympathetic nerve hyperexcitation in the early clinical stage of AD

[0077] (1) Transcriptome sequencing analysis and verification:

[0078] Norepinephrine (NE), the most important neurotransmitter of the sympathetic nerves, was used to simulate an in vitro environment of excessive nerve excitation. Endothelial cells were stimulated with NE at different concentrations of 0.01-400 μM for 48 hours, and the appropriate concentration was screened by CCK8. After stimulating the cells with 10 μM NE for 48 hours, transcriptome sequencing detection and analysis were performed. The sample pretreatment steps are as follows: wash the stimulated cells with pre-cooled 1× PBS; add an appropriate amount of lysis buffer (TRIzol) and use a pipette to blow thoroughly to fully lyse the cells; transfer the lysate to a 1.5 mL enzyme-free tube and store at -80°C; send with sufficient dry ice. Subsequent operations were completed by "Ouyi Bio". The results of preliminary sequencing analysis showed that endothelial cells mainly showed changes such as cell cycle arrest, activation of the P53 signaling pathway and cell senescence.

[0079] (2) Verification of endothelial cell senescence phenotype

[0080] a. In vitro validation: Cells were treated with 100 μM H2O2 as a positive control and stimulated with 10 μM and 100 μM NE for 48 hours. RNA and protein were extracted, and changes in endothelial cell senescence markers, such as P53, P21, and P16, were detected using qRT-PCR and western blot. Cell senescence was assessed using β-galactosidase (β-gal) staining and HMGB1 immunofluorescence staining.

[0081] b. In vivo validation: Frozen sections were obtained from the blank control (WT), experimental control (APP / PS1), propranolol-treated (APP / PS1+PRO), and 6-hydroxydopamine-treated (APP / PS1+6-OHDA) groups and immunostained for CD31 and β-gal to assess senescence of H-type vascular endothelial cells in the jaws and long bones. Simultaneously, bone H-type vascular endothelial cells were isolated using the same method as in Part 1. RNA was extracted from cells cultured to the third passage, and qRT-PCR was used to detect changes in senescence genes, including P53, P21, and P16.

[0082] 2. Clarify the pathway by which senescence of H-type vascular endothelial cells induces vascular osteogenesis impairment in the preclinical phase of AD

[0083] (1) Bioinformatics screening and analysis:

[0084] In-depth analysis of the sequencing results revealed that endothelial cells exhibited typical mitochondrial dysfunction-induced senescence (MiDAS), including: high p53, low NF-kB, low HMGB1, lack of IL-1β-dependent inflammatory arm, low LMNB1, and cell arrest.

[0085] (2) In vitro verification of MiDAS in endothelial cells:

[0086] a. Cells were divided into two groups: a blank control group and a 10 μM NE group. The expression levels of inflammatory factors such as IL-1β, IL-6, and TNF-α in the supernatant were measured using ELISA. Changes in LMNB1 expression were detected using western blot and immunofluorescence staining. The cell cycle status of endothelial cells in each group was analyzed using flow cytometry. A low NAD+ / NADH ratio is a typical characteristic of MiDAS. Changes in the NAD+ / NADH ratio in endothelial cells after NE treatment were measured using a kit.

[0087] b. In vitro rescue experiment: β-Nicotinamide mononucleotide (NMN) is a key exogenous NAD+ intermediate. MiDAS rescue experiments were performed using 500 μM NMN. Cells were divided into two groups: a NE control group and a NE+NMN group. Cell senescence was assessed by qRT-PCR and western blot analysis to detect changes in endothelial cell factors such as P53, P21, and P16. β-gal staining, HMGB1, and LMNB1 fluorescence staining, and flow cytometry were performed to analyze the cell cycle.

[0088] (3) Detection of angiogenic-osteogenic coupling factors:

[0089] a. MiDAS is characterized by the lack of an inflammatory arm, namely, decreased expression of the senescence-associated secretory phenotype (SASP). ELISA was used to detect the expression levels of inflammatory factors such as IL-1β, IL-6, and TNF-α in the supernatants of endothelial cells from the NE group and the NE+NMN group. To further clarify the cause of the imbalance in angiogenesis-osteogenesis coupling, ELISA was used to detect changes in the expression of specific angiogenesis-osteogenesis coupling factors (Noggin, TGF-β, etc.) in the supernatants of the blank control group, the NE group, and the NE+NMN group to clarify the possible pathways by which NE-induced MiDAS in vascular endothelial cells leads to vascular osteogenesis disorders.

[0090] b. Vascular osteogenesis function test: Take the supernatant of the blank control group, NE group, and NE+NMN group, mix it with the standard osteogenic induction solution in a ratio of 1:1 to prepare osteogenic induction solution. The subsequent processing and testing are the same as those in Part 1, 2-(4).

[0091] 3: Exploring the mechanism by which sympathetic nerve excitation leads to senescence of bone H-type vascular endothelial cells

[0092] 1. Screen and verify the key role of PKM2-mediated glycolysis abnormalities in sympathetic nerve hyperexcitation driving MiDAS in H-type vascular endothelial cells

[0093] (1) Transcriptomics data screening and in vitro validation:

[0094] a. KEGG and GSEA enrichment revealed significant energy metabolism abnormalities in vascular endothelial cells. Cells were stimulated with 10μM and 100μM NE for 48 hours, and flow cytometry was used to measure changes in endothelial cell uptake of a fluorescently labeled 2-deoxyglucose analog (2-NDBG). Deep transcriptomic data were mined to identify genetic differences in enzymes involved in glucose metabolism, identifying pyruvate kinase (PK), a key enzyme in glycolysis.

[0095] b. qRT-PCR was used in vitro to confirm that vascular endothelial cells primarily express the M2 pyruvate kinase (PKM2). Cells were stimulated with 10 μM NE for 48 hours, and changes in PKM2 expression in vascular endothelial cells were detected using qRT-PCR and western blot. Immunofluorescence staining was used to observe changes in the distribution and expression levels of PKM2 in endothelial cells. PKM2 activity was measured using a PK activity assay kit, pyruvate content was measured using a pyruvate content assay kit, lactate content was measured using an L-lactic acid content assay kit, and ATP content was measured using an ATP content assay kit to clarify changes in PKM2 function.

[0096] (2) Verification of PKM2 Control of Endothelial Cell Senescence:

[0097] a. Stimulate endothelial cells with the PKM2 inhibitor Compound 3k (50 μM) for 48 hours and measure PKM2 activity using a PK activity assay kit. Quantitative real-time PCR (qRT-PCR) and western blot were used to examine changes in endothelial cell senescence markers, including P53, P21, P16, LMNB1, and HMGB1-related factors, as well as β-gal staining and HMGB1 immunofluorescence staining, to clarify the key role of PKM2 in MiDAS in endothelial cells.

[0098] b. NE-treated endothelial cells were rescued using the PKM2 promoter TEPP-46 (100 nM). PKM2 activity, pyruvate, lactate, and ATP levels were measured using relevant kits. Changes in endothelial cell senescence markers, including P53, P21, P16, LMNB1, and HMGB1, were detected using qRT-PCR and western blot, as well as β-gal staining and HMGB1 immunofluorescence staining. Furthermore, ELISA was used to examine the expression of factors such as Noggin, an angiogenic-osteogenic coupling factor, and TGF-β in the supernatant of rescued endothelial cells.

[0099] (3) Vascular osteogenesis function test:

[0100] The supernatant of the NE group and the NE+TEPP-46 group was mixed with the standard osteogenic induction solution at a ratio of 1:1 to prepare the osteogenic induction solution. Subsequent processing and testing were the same as in Part 1, 2-(4).

[0101] (4) In vivo experimental verification:

[0102] We designed and constructed an AAV virus that specifically overexpresses PKM2 in vascular endothelial cells. Ten 3-month-old male APP / PS1 mice were selected and the constructed AAV-PKM2 virus was specifically introduced into the mandibular condyle and tibial H-type vascular endothelial cells using intramedullary injection. Four weeks later, the mice were harvested. The mandibular and leg bones were isolated. Specific processing and testing were the same as in Part 1, Section 2-(2).

[0103] 2. The transcription factor c-MAF is essential for regulating PKM2 expression and activity to control MiDAS in endothelial cells

[0104] (1) Screening of transcription factors:

[0105] Transcriptome analysis revealed significant changes in the transcription factor c-MAF, ranking among the top five genes. Cells were stimulated with 10 μM NE for 48 hours in vitro, and c-MAF expression was assessed using qRT-PCR, western blot, and immunofluorescence. Potential binding sites for c-MAF were predicted within the PKM2 gene promoter and nearby regions using the NCBI and JASPAR databases.

[0106] (2) In vitro experimental verification:

[0107] ChIP-qPCR and electrophoretic mobility shift assays (EMSA) were used to verify the direct binding of c-MAF to specific sequences in the PKM2 gene. Furthermore, by constructing c-MAF mutants and conducting PKM2 luciferase reporter assays, key domains regulating PKM2 transcriptional activation or repression were identified. At the cellular level, c-MAF was overexpressed using siRNA or plasmids / viruses, and changes in PKM2 expression and activity, glycolysis products, ATP production, membrane potential, and other functional indicators were measured to confirm that c-MAF is a key transcription factor regulating PKM2 expression and activity.

[0108] (3) Verify that c-MAF regulates PKM2-mediated endothelial cell senescence and vascular osteogenesis disorders: Overexpress c-MAF in endothelial cells using plasmids or lentivirus, and detect changes in endothelial cell senescence indicators using qRT-PCR, western blot, and other methods. Use ELISA to detect changes in the expression of osteoblast-angiogenic coupling factors Noggin and TGF-β in vascular endothelial cells. Take the supernatant and mix it with standard osteogenic induction solution in a 1:1 ratio to prepare osteogenic induction solution. Subsequent processing and detection are the same as in Part 1, 2-(4).

[0109] 4. Exploring the synergistic effect of ED-71 and oryzanol on vascular osteogenesis disorders in the early clinical stages of AD

[0110] (1) Animal model establishment and processing:

[0111] Three-month-old male APP / PS1 mice were selected as a preclinical model of AD. The mice were divided into the following groups: experimental control group (APP / PS1), oryzanol-treated group (APP / PS1+ORZ), ED-71-treated group (APP / PS1+ED-71), and oryzanol and ED-71-treated group (APP / PS1+ORZ+ED-71). There were 10 mice in each group. Oral administration of 100 mg / kg of oryzanol (ORZ) was performed once a day; and oral administration of 50 ng / kg of ED-71 was performed once a day. After 4 weeks of drug treatment, blood was collected from the anesthetized mice's eyeballs, and the serum was separated by centrifugation at 3000 rpm / min for 10 minutes. The serum was stored at -80°C and used for ELISA to detect changes in plasma NE content. After the mice were sacrificed, the jaws and leg bones were isolated.

[0112] (2) Tissue processing and related testing:

[0113] The isolated mandibles and tibiae were fixed, decalcified, dehydrated, embedded, and sectioned, and paraffin and frozen sections were prepared. Paraffin sections were stained with hematoxylin and eosin (HE) and Masson staining to analyze morphological changes in the mandibles and tibiae of each group. Immunohistochemistry was also used to detect osteoblastic markers, such as alkaline phosphatase (ALP) and runx2. Frozen sections were evaluated for the presence of H-type vessels using immunofluorescence staining for CD31 and EMCN; for sympathetic nerve function using immunofluorescence staining for CD31 and TH; for endothelial cell senescence using immunofluorescence staining for CD31 and β-gal; and for changes in the number of osteoprogenitor cells surrounding H-type vessels using immunofluorescence staining for CD31 and osterix. Additionally, changes in bone mass were analyzed using micro-computed tomography (MicroCT). Five mice were randomly selected from each group and received a local injection of 8 mg / kg of calcein into the leg muscle on the 13th and 3rd day before the end of drug treatment. The samples were collected in the dark, and hard tissue sections were made to detect the rate of new bone formation to evaluate the improvement effects of ORZ and ED-71 on H-type blood vessels and vascular osteogenesis in preclinical AD mice.

[0114] Experimental results

[0115] 1. Sympathetic nerve overactivation is associated with impaired vascularized bone formation in preclinical Alzheimer's disease mice

[0116] To evaluate the vascularized bone formation capacity in preclinical Alzheimer's disease mice, we analyzed 3- and 4-month-old APP / PS1 mice and wild-type mice. Figure 1 As shown in Figure a, the density of H-type blood vessels near the growth plate of 3- and 4-month-old APP / PS1 mice was significantly reduced compared with wild-type mice of the same age. Flow cytometric analysis showed that the density of H-type endothelial cells in the femur of APP / PS1 mice was also significantly lower than that of wild-type mice of the same age. Given our previous findings that Aβ damages H-type blood vessels, we examined Aβ levels in bone vessels and found no significant difference between APP / PS1 mice and controls. ELISA assays for norepinephrine (NE) levels in bone tissue and serum revealed significantly increased NE levels in APP / PS1 mice. Immunofluorescence staining also confirmed an increase in sympathetic nerves around H-type blood vessels in APP / PS1 mice, indicating enhanced sympathetic nerve activity. These results suggest that sympathetic overactivity is associated with impaired vascularized bone formation in preclinical Alzheimer's disease mice.

[0117] 2. Inhibiting sympathetic nerve overactivation can reverse the impairment of vascularized bone formation in preclinical Alzheimer's disease mice

[0118] To investigate whether sympathetic nerve overactivation leads to impaired vascularized osteogenesis, we used propranolol or 6-hydroxydopamine (6-OHDA) to reduce sympathetic nerve activity in APP / PS1 mice. After treatment, CD31hiEMCNhi staining and the number of Osterix+ osteoblast progenitor cells were significantly restored. ALP and RUNX2 immunohistochemistry revealed enhanced osteoblast activity, and micro-CT analysis revealed improved trabecular bone structure and increased bone volume. Bone endothelial cells sorted by MACS-EMCN secreted elevated levels of Noggin and TGF-β, and their conditioned medium significantly promoted ALP activity and calcium deposition in osteoblast progenitor cells. These results suggest that inhibiting sympathetic nerve overactivation can effectively reverse the impairment of vascularized osteogenesis.

[0119] 3. Sympathetic nerve overactivation impairs vascularized osteogenesis by promoting senescence of H-type endothelial cells

[0120] In vitro, treatment with pathological concentrations of NE resulted in the upregulation of 734 genes and the downregulation of 550 genes in vascular endothelial cells. KEGG analysis revealed significant enrichment of cell cycle, p53 signaling, and cellular senescence pathways. Dose-dependent experiments confirmed that NE upregulated the expression of senescence-related factors, increased the proportion of β-galactosidase-positive cells, and led to nuclear HMGB1 loss. Treatment with propranolol and 6-OHDA reduced the number of CD31+β-gal+ cells and decreased the expression of p16, p21, and p53 in bone tissue. Mitochondrial dysfunction-associated senescence (MiDAS) analysis revealed that NE-treated cells exhibited characteristics of the inflammatory axis, including high p53, low NF-κB, and lack of IL-1β. Supplementation with β-nicotinamide mononucleotide (NMN) reversed mitochondrial dysfunction and cellular senescence, restoring vascularized bone formation.

[0121] PKM2-mediated glycolysis is key to sympathetic nerve overactivation-induced senescence in H-type endothelial cells

[0122] Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed significant alterations in carbohydrate metabolism pathways in NE-treated cells. 2-NBDG uptake experiments revealed decreased glucose utilization and suppressed pyruvate kinase activity, with PKM2 being the primary regulatory isoform. NE treatment resulted in decreased PKM2 expression and activity, an effect reversed by the β2-adrenergic receptor antagonist ICI-118551. Activation of PKM2 (TEPP-46) or pyruvate supplementation reduced the proportion of senescent cells and restored osteogenic factor secretion and calcium deposition. In vivo, AAV-PKM2 overexpression or pyruvate supplementation significantly ameliorated bone vascular senescence and bone loss in APP / PS1 mice.

[0123] c-Maf participates in the regulation of PKM2-mediated glycolysis in NE-treated vascular endothelial cells

[0124] Differential gene expression analysis revealed that c-maf was significantly downregulated in the NE-treated group, an effect reversed by ICI-118551. Bioinformatics prediction and dual-luciferase reporter assays confirmed that c-Maf directly binds to the PKM2 promoter. Overexpression of c-Maf restored PKM2 expression, enhanced glycolytic flux, and reduced cell senescence. Inhibition of c-Maf (Nivalenol) produced the opposite effect. Overexpression of c-Maf also restored the osteogenic induction capacity of NE-treated endothelial cells.

[0125] 6. Oryzanol and ED-71 synergistically improve vascularized osteogenesis impairment in Alzheimer's disease mice

[0126] Combination therapy experiments showed that oryzanol reduced NE levels, while ED-71 had no such effect. However, both agents reduced the proportion of senescent cells, with the combined treatment being more effective. They also synergistically increased the number of Osterix+ osteoblast progenitor cells and enhanced mineralized bone formation. HE staining and micro-CT analysis confirmed that combined treatment significantly improved bone density and structure.

[0127] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application. Various modifications and variations are possible for those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present application shall be included within the scope of protection of the present application.

Claims

1. Application of oryzanol combined with ED-71 in the preparation of drugs for preventing and / or treating vascular osteogenesis disorder in the preclinical stage of Alzheimer's disease.

2. The use according to claim 1, characterized in that Specifically, the prevention and / or treatment of vascular osteogenesis disorder in the preclinical stage of Alzheimer's disease is specifically manifested by any one or more of the following (a)-(f): (a) Reduce the expression level of norepinephrine; (b) inhibiting sympathetic nerve overactivation-induced H-type endothelial cell senescence; (c) increase the number of Osterix+ osteoblast progenitor cells; (d) enhance mineralized bone formation; (e) Improve bone density and structure; (f) Improve the impairment of vascularized bone formation in the preclinical stage of Alzheimer's disease.

3. The use according to claim 1 or 2, characterized in that When the oryzanol and ED-71 are used together, the mass ratio of the two is 0.5-5×10 6 :

1.

4. A pharmaceutical composition for preventing and / or treating vascular osteogenesis disorder in the preclinical stage of Alzheimer's disease, characterized in that: The active ingredients of the pharmaceutical composition at least contain oryzanol and ED-71.

5. The pharmaceutical composition according to claim 4, wherein The mass ratio of oryzanol to ED-71 is 0.5-5×10 6 :

1.

6. The pharmaceutical composition according to claim 5, wherein The pharmaceutical composition further comprises at least one non-pharmaceutically active ingredient.

7. The pharmaceutical composition according to claim 6, wherein The inactive ingredients of the medicine are carriers, excipients and diluents commonly used in pharmacy.

8. The pharmaceutical composition according to claim 6, wherein The dosage form of the pharmaceutical composition is powder, granule, suspension, emulsion, syrup, spray oral preparation, external preparation, suppository and sterile injection solution.

9. The pharmaceutical composition according to claim 6, wherein The subject of drug administration is a human or a non-human mammal.

10. The pharmaceutical composition according to any one of claims 4 to 9, characterized in that The prevention and / or treatment of preclinical vascular osteogenesis disorder of Alzheimer's disease is specifically manifested in any one or more of the following (a)-(f): (a) Reduce the expression level of norepinephrine; (b) inhibiting sympathetic nerve overactivation-induced H-type endothelial cell senescence; (c) increase the number of Osterix+ osteoblast progenitor cells; (d) enhance mineralized bone formation; (e) Improve bone density and structure; (f) Improve the impairment of vascularized bone formation in the preclinical stage of Alzheimer's disease.

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