Application of insulin and analogue thereof in preparation of medicine for preventing or treating Alzheimer's disease

Abstract

The invention relates to application of insulin and analogues thereof in preparation of medicines for preventing or treating Alzheimer's disease. Researches find that the damage effect of A beta on insulin secretion depends on CX3CR1, the blood plasma insulin level of AD model mice can be recovered by supplementing exogenous insulin, and the A beta has the treatment effects of improving cognitive impairment of AD model mice, improving cerebrovascular lesion, recovering brain glycometabolism function, relieving brain inflammatory response and the like; the invention provides an alternative medicine with definite curative effect, high safety and affordable price for Alzheimer's disease, expands the indications of insulin and analogues thereof, and has wide market prospect and important social significance.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the use of insulin and its analogues in the preparation of drugs for the prevention or treatment of Alzheimer's disease. Background Technology

[0002] Alzheimer's disease (AD) is a neurodegenerative disease caused by the death of nerve cells in the brain. It is the most common cause of dementia, accounting for approximately 60-80% of all dementia cases in the elderly. As AD progresses, patients gradually lose memory and cognitive abilities, severely impacting their daily activities and social life, placing a heavy burden on families and society. According to data from *The Lancet Public Health*, the number of people with dementia worldwide was estimated at 57.4 million in 2019, and is projected to increase to 83.2 million by 2030, 116 million by 2040, and 152.8 million by 2050. The *China Alzheimer's Disease Report 2024* shows that in 2021, the number of people with AD and other dementias in my country reached 17 million. According to the World Health Organization (WHO), the global cost of treating dementia was estimated at US$1.3 trillion in 2019 and is projected to increase to US$2.3 trillion by 2030, potentially disrupting global social and economic development and overwhelming health and social service systems. Data shows that the average annual cost per patient is US$15,889 for mild dementia, US$26,859 for moderate dementia, and US$36,180 for severe dementia. In 2015, the average annual cost per Alzheimer's patient in my country reached 130,000 yuan, with over 67% of that going towards indirect medical expenses. The new Alzheimer's drug, lencanezumab injection, approved for marketing in my country in 2024, has an annual treatment cost of approximately 180,000 yuan.

[0003] The pathogenesis of Alzheimer's disease is complex, primarily involving the amyloid β-protein (Aβ) hypothesis, the abnormal phosphorylation of Tao protein, central cholinergic system damage, inflammatory damage, excitatory nerve damage, and gut microbiota theory. Among these, Aβ protein deposition and Tau neurofibrillary tangles are considered major mechanisms of AD pathogenesis. Clinically, various drugs are commonly used to control dementia symptoms, including cholinesterase inhibitors such as donepezil and NMDA receptor antagonists such as methimazole. However, the number of drugs available for controlling AD symptoms is very limited, and there is currently no cure for AD. Therefore, researching and developing novel, effective, and affordable AD treatments is of great significance. Summary of the Invention

[0004] This application found through research that Aβ has a damaging effect on insulin secretion and that this damaging effect is dependent on CX3CR1. Supplementing with exogenous insulin can restore plasma insulin levels in AD model mice and has therapeutic effects such as improving cognitive dysfunction, improving cerebrovascular lesions, restoring brain glucose metabolism function, and reducing brain inflammation in AD model mice.

[0005] Based on this, the first aspect of this application provides the use of insulin and its analogues in the preparation of medicaments for the prevention or treatment of Alzheimer's disease.

[0006] The second aspect of this application provides the use of insulin and its analogues in the preparation of medicaments that increase plasma insulin levels in Alzheimer's disease.

[0007] The third aspect of this application provides the use of insulin and its analogues in the preparation of medicaments that improve cognitive impairment in Alzheimer's disease.

[0008] The fourth aspect of this application provides the use of insulin and its analogues in the preparation of medicaments for improving microvascular lesions in the brain of Alzheimer's disease.

[0009] The fifth aspect of this application provides the use of insulin and its analogues in the preparation of medicaments for restoring brain glucose metabolism function in Alzheimer's disease.

[0010] The sixth aspect of this application provides the use of insulin and its analogues in the preparation of medicaments that reduce the inflammatory response in Alzheimer's disease.

[0011] In some embodiments, the insulin includes at least one of human insulin, glargine insulin, aspart insulin, lispro insulin, glutathione insulin, detemir insulin, and degludec insulin.

[0012] In some embodiments, the administration method of the drug includes intravenous injection, intraperitoneal injection, and subcutaneous injection.

[0013] In some embodiments, the dosage of the insulin and its analogues is 0.5 U / kg to 2 U / kg.

[0014] The seventh embodiment of this application provides a drug that can prevent or treat Alzheimer's disease, and / or increase plasma insulin levels in Alzheimer's disease, and / or improve cognitive impairment in Alzheimer's disease, and / or improve microvascular lesions in the brain in Alzheimer's disease, and / or reduce inflammatory responses in Alzheimer's disease, wherein the drug includes insulin and its analogues.

[0015] In some embodiments, the insulin includes at least one of human insulin, glargine insulin, aspart insulin, lispro insulin, glutathione insulin, detemir insulin, and degludec insulin.

[0016] In some embodiments, the drug further includes one or more pharmaceutically acceptable excipients.

[0017] This application describes the preparation of insulin and its analogues as drugs for the treatment of Alzheimer's disease. Experimental verification has shown that insulin and its analogues have great potential as drugs for the prevention and treatment of Alzheimer's disease, and have a good therapeutic effect on Alzheimer's disease. This provides a drug alternative with definite efficacy, high safety and affordability for the prevention and treatment of Alzheimer's disease, expands the indications of insulin and its analogues, and has broad market prospects and important social significance. Attached Figure Description

[0018] Figure 1 The impaired effect of Aβ on pancreatic insulin secretion (in Aβ-induced AD model mice); Figure 2 The damaging effect of Aβ on pancreatic insulin secretion (in 5xFAD transgenic AD model mice); Figure 3 The damaging effect of Aβ on pancreatic insulin secretion (in AD patients); Figure 4 The damaging effect of Aβ on pancreatic secretion is dependent on CX3CR1 (experimental study of silencing CX3CR1 expression in the pancreas by in situ injection of LV viral vector). Figure 5 The damaging effect of Aβ on pancreatic secretion is dependent on CX3CR1 (conditional knockout of CX3CR1 in transgenic mice). Figure 6 The effect of exogenous insulin intake on plasma insulin and glucose levels in AD model mice (in Aβ-induced AD model mice). Figure 7 The effect of exogenous insulin intake on cognitive impairment in AD model mice (in Aβ-induced AD model mice). Figure 8 The effect of exogenous insulin intake on brain glucose metabolism in AD model mice (in Aβ-induced AD model mice). Figure 9 The effect of exogenous insulin intake on vascular structure and function in AD model mice (in Aβ-induced AD model mice). Figure 10 The effect of exogenous insulin intake on plasma insulin and glucose levels in AD model mice (in 5xFAD transgenic AD model mice). Figure 11 The effect of exogenous insulin intake on cognitive impairment in AD model mice (in 5xFAD transgenic AD model mice). Figure 12The effect of exogenous insulin intake on brain glucose metabolism in AD model mice (in 5xFAD transgenic AD model mice). Figure 13 The effect of exogenous insulin intake on vascular structure and function in AD model mice (in 5xFAD transgenic AD model mice). Figure 14 Effects of exogenous insulin intake on the whole brain microvascular system in AD model mice (3D fluorescence imaging of whole brain microvessels labeled with Lectin) (in 5xFAD transgenic AD model mice); Figure 15 Effects of exogenous insulin intake on cerebral blood flow signals in AD model mice (color Doppler blood flow imaging) (in 5xFAD transgenic AD model mice); Figure 16 The effect of exogenous insulin intake on serum inflammatory factors in AD model mice (in 5xFAD transgenic AD model mice); Figure 17 The effect of exogenous insulin intake on astrocytes in the brain of AD model mice (in 5xFAD transgenic AD model mice). Figure 18 The effect of exogenous insulin intake on microglia in the brain of AD model mice (in 5xFAD transgenic AD model mice). Figure 19 The effect of exogenous insulin intake on Aβ deposition in the brains of AD model mice (in 5xFAD transgenic AD model mice); Figure 20 The effects of insulin on the expression of FOXO3, pAKT2, AKT2, GLUT1, HIF1α and VEGF proteins in hCMEC / D3 cells; Figure 21 The effects of insulin on the levels of Akt1, Akt3, FOXO4 and INSR secreted by hCMEC / D3 cells; Figure 22 To investigate the therapeutic effects of different doses of insulin on 5xFAD transgenic AD model mice. Detailed Implementation

[0019] The present application will be further described in detail below with reference to the embodiments and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Furthermore, numerous specific details are set forth in the following description to provide a more complete understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0020] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in this application and in its specification is for descriptive purposes only and is not intended to be limiting of the application.

[0021] Unless otherwise stated or in case of conflict, the terms or phrases used in this application shall have the following meanings: The terms "and / or," "or / and," and "and / or" as used in this application encompass any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected using at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes solutions connected by "logical AND," and also undoubtedly includes solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0022] In this application, terms such as "preferred," "better," "more suitable," and "ideal" are merely used to describe implementation methods or embodiments that achieve better results, and should be understood not to limit the scope of protection of this application.

[0023] In this application, terms such as "further," "even more," and "particularly" are used for descriptive purposes and to indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0024] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0025] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0026] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0027] In this application, %(w / w) and wt% both represent weight percentage, %(v / v) refers to volume percentage, and %(w / v) refers to mass-volume percentage.

[0028] All references to this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the purpose and / or technical solution of this application, all cited references are incorporated herein by reference in their entirety and for all purposes. When references are cited in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. Examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0029] With the increasing aging of the global population, the number of Alzheimer's disease patients is rapidly increasing, making Alzheimer's disease a global public health problem. Because the pathogenesis of Alzheimer's disease is unclear, progress in the development of anti-AD drugs is slow, but research and development of AD drugs has not stopped both domestically and internationally. To date, no specific drug for Alzheimer's disease has been developed globally. Clinically, limited drugs such as donepezil and memantine can only help control AD ​​symptoms, and there is a need to develop better symptomatic AD drugs to meet the needs of different AD patients.

[0030] This application found through research that Aβ has a damaging effect on insulin secretion and that this damaging effect is dependent on CX3CR1. Supplementing with exogenous insulin can restore plasma insulin levels in AD model mice and has therapeutic effects such as improving cognitive dysfunction, improving cerebrovascular lesions, restoring brain glucose metabolism function, and reducing brain inflammation in AD model mice.

[0031] Based on this, the first aspect of this application provides the use of insulin and its analogues in the preparation of medicaments for the prevention or treatment of Alzheimer's disease.

[0032] This application describes the formulation of insulin and its analogues as drugs for treating Alzheimer's disease. Insulin and its analogues compensate for the reduction in pancreatic insulin secretion caused by Aβ by supplementing the body's insulin levels. By taking in insulin from exogenous sources, they restore the body's insulin levels and microenvironment, thereby restoring plasma insulin levels in AD model mice. They have therapeutic effects such as improving cognitive dysfunction, improving cerebrovascular lesions, restoring brain glucose metabolism function, and reducing brain inflammation in AD model mice. This provides a definite, safe, and affordable alternative drug for the prevention and treatment of Alzheimer's disease, expands the indications for insulin and its analogues, and has broad market prospects and important social significance.

[0033] In some embodiments, the insulin includes at least one of human insulin, glargine insulin, aspart insulin, lispro insulin, glutathione insulin, detemir insulin, and degludec insulin.

[0034] In some embodiments, the administration method of the drug includes intravenous injection, intraperitoneal injection, and subcutaneous injection.

[0035] In some embodiments, the dosage of the insulin and its analogues is 0.5 U / kg to 2 U / kg. This dosage is beneficial for the prevention or treatment of Alzheimer's disease.

[0036] The second aspect of this application provides the use of insulin and its analogues in the preparation of medicaments that increase plasma insulin levels in Alzheimer's disease.

[0037] This study found that supplementing with exogenous insulin can restore plasma insulin levels in mice in the Aβ-induced AD model group. Therefore, insulin and its analogues can be used to prepare drugs that improve plasma insulin levels in Alzheimer's disease.

[0038] In some embodiments, the insulin includes at least one of human insulin, glargine insulin, aspart insulin, lispro insulin, glutathione insulin, detemir insulin, and degludec insulin.

[0039] In some embodiments, the administration method of the drug includes intravenous injection, intraperitoneal injection, and subcutaneous injection.

[0040] In some embodiments, the dosage of the insulin and its analogues is 0.5 U / kg to 2 U / kg. This dosage is beneficial for increasing plasma insulin levels in Alzheimer's patients.

[0041] The third aspect of this application provides the use of insulin and its analogues in the preparation of medicaments that improve cognitive impairment in Alzheimer's disease.

[0042] This study found that supplementing with exogenous insulin can improve cognitive and memory impairment in mice with Aβ-induced AD. Therefore, insulin and its analogues can be used to prepare drugs that improve cognitive impairment in Alzheimer's disease.

[0043] In some embodiments, the insulin includes at least one of human insulin, glargine insulin, aspart insulin, lispro insulin, glutathione insulin, detemir insulin, and degludec insulin.

[0044] In some embodiments, the administration method of the drug includes intravenous injection, intraperitoneal injection, and subcutaneous injection.

[0045] In some embodiments, the dosage of the insulin and its analogues is 0.5 U / kg to 2 U / kg. This dosage is beneficial in improving cognitive impairment in Alzheimer's disease.

[0046] The fourth aspect of this application provides the use of insulin and its analogues in the preparation of medicaments for improving microvascular lesions in the brain of Alzheimer's disease.

[0047] This study found that supplementing with exogenous insulin can restore the brain microvascular structure and cerebral blood flow in 5xFAD transgenic AD model mice. Furthermore, insulin also regulates the insulin signaling pathway in human brain microvascular endothelial cells through AKT2 / FOXO3. Therefore, insulin and its analogues can be used to prepare drugs that improve brain microvascular lesions in Alzheimer's disease.

[0048] In some embodiments, the insulin includes at least one of human insulin, glargine insulin, aspart insulin, lispro insulin, glutathione insulin, detemir insulin, and degludec insulin.

[0049] In some embodiments, the administration method of the drug includes intravenous injection, intraperitoneal injection, and subcutaneous injection.

[0050] In some embodiments, the dosage of the insulin and its analogues is 0.5 U / kg to 2 U / kg. This dosage is beneficial in improving microvascular lesions in the brain caused by Alzheimer's disease.

[0051] The fifth aspect of this application provides the use of insulin and its analogues in the preparation of medicaments for restoring brain glucose metabolism function in Alzheimer's disease.

[0052] This study found that supplementing with exogenous insulin can increase the uptake and utilization of glucose in the brains of 5xFAD transgenic AD model mice, and improve the glucose utilization rate in the brains of 5xFAD transgenic AD model mice. Therefore, insulin and its analogues can be used to prepare drugs that restore brain glucose metabolism function in Alzheimer's disease.

[0053] In some embodiments, the insulin includes at least one of human insulin, glargine insulin, aspart insulin, lispro insulin, glutathione insulin, detemir insulin, and degludec insulin.

[0054] In some embodiments, the administration method of the drug includes intravenous injection, intraperitoneal injection, and subcutaneous injection.

[0055] In some embodiments, the dosage of the insulin and its analogues is 0.5 U / kg to 2 U / kg. This dosage is beneficial for restoring brain glucose metabolism function in Alzheimer's disease.

[0056] The sixth aspect of this application provides the use of insulin and its analogues in the preparation of medicaments that reduce the inflammatory response in Alzheimer's disease.

[0057] This study found that serum levels of cytokines such as IL-13, GM-CSF, IL-2, IL-1β, and TNF-α were reduced in mice (5xFAD-Insulin group) after exogenous insulin supplementation, and IL-1α and IL-10 cytokines were also reduced. This indicates that exogenous insulin supplementation can improve the inflammatory response in 5xFAD transgenic AD model mice. Therefore, insulin and its analogues can be used to prepare drugs that alleviate the inflammatory response in Alzheimer's disease.

[0058] In some embodiments, the insulin includes at least one of human insulin, glargine insulin, aspart insulin, lispro insulin, glutathione insulin, detemir insulin, and degludec insulin.

[0059] In some embodiments, the administration method of the drug includes intravenous injection, intraperitoneal injection, and subcutaneous injection.

[0060] In some embodiments, the dosage of the insulin and its analogues is 0.5 U / kg to 2 U / kg. This dosage is beneficial in reducing the inflammatory response in Alzheimer's disease.

[0061] The seventh aspect of this application provides a medicament capable of preventing or treating Alzheimer's disease, and / or increasing plasma insulin levels in Alzheimer's disease, and / or improving cognitive impairment in Alzheimer's disease, and / or improving microvascular lesions in the brain in Alzheimer's disease, and / or reducing inflammatory responses in Alzheimer's disease, said medicament comprising insulin and analogues thereof.

[0062] In some embodiments, the insulin includes at least one of human insulin, glargine insulin, aspart insulin, lispro insulin, glutathione insulin, detemir insulin, and degludec insulin.

[0063] In some embodiments, the drug further includes one or more pharmaceutically acceptable excipients.

[0064] This application applies insulin and its analogues to prevent or treat Alzheimer's disease, and / or increase plasma insulin levels in Alzheimer's patients, and / or improve cognitive impairment in Alzheimer's patients, and / or improve microvascular lesions in the brain in Alzheimer's patients, and / or reduce inflammatory responses in Alzheimer's patients, providing a new treatment strategy for the prevention and treatment of Alzheimer's disease.

[0065] The following is a specific embodiment.

[0066] Unless otherwise specified, the reagents and instruments used in the examples are conventionally selected in the art. Experimental methods not specifying particular conditions in the examples are typically performed under standard conditions, such as those described in literature, books, or recommended by the reagent kit manufacturer. All reagents used in the examples are commercially available.

[0067] Unless otherwise specified, the experimental methods in the following examples are as follows: Experimental methods: 1. Laboratory animals 5xFAD transgenic mice were derived from the Jackson Laboratory in the United States (catalog number: 34348), Pdx Cre Transgenic mice and CX3CR1 loxp / loxp Transgenic mice were purchased from Cyagen (Suzhou) Biotechnology Co., Ltd., and C57BL / 6 mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0068] All animals were housed in an SPF-grade animal facility. The temperature in the animal facility was controlled at 22℃ ± 2℃, and the humidity was controlled at 60%~80%. Mice were allowed free access to standard feed, and there was a 12-hour light / dark cycle.

[0069] 2. Alzheimer's disease patient cohort This study included AD patients (>50 years old and without diabetes) hospitalized between January 2023 and June 2024. Inclusion criteria: ① Meeting the diagnostic criteria for AD; ② Age >50 years and hospitalization duration >2 years; ③ Informed consent signed by the patient and their family. Exclusion criteria: ① Severe organ failure; ② Terminally ill patients; ③ Poor treatment adherence; ④ Incomplete clinical data; ⑤ Medication history could not be obtained within 48 hours of admission.

[0070] Fasting venous blood was collected from patients (AD group) and age-matched controls (Non-AD group) after 2 hours. Plasma insulin levels were measured using an insulin radioimmunoassay kit (brand: Beijing Northern Biotechnology Research Institute, catalog number: S20083079) and the readings were obtained from the enzyme-linked immunosorbent assay (ELISA) reader.

[0071] 3. Preparation of Aβ oligomers 1 mg of human Aβ42 (brand: AnaSpec, catalog number: AS-24224) was dissolved in pre-chilled 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP, brand: aldaddin, catalog number: H07503), vortexed for 30 seconds, and incubated at room temperature for 2 hours for monomerization. After drying in a fume hood, it was stored at -80°C. The dried Aβ was reconstituted with DMSO to 5 mM, sonicated at room temperature for 10 min, and then diluted with PBS to 100 μM. After oligomerization by standing at 4°C for 12 hours, it was stored at -80°C.

[0072] 4. Construct a lentivirus-mediated vector targeting CX3CR1 gene knockout. CRISPR targets were designed targeting exon 2 of the mouse CX3CR1 gene. sgRNA sequences were screened using an online design platform (https: / / benchling.com). The forward and reverse primer sequences were 5'-CACCGTGTCTGGGTGACTACCCCG-3' (as shown in SEQ ID NO.1) and 5'-AAACCGGGGTAGTCACCCAGACAC-3' (as shown in SEQ ID NO.2), respectively. Lentiviral vectors (sgCX3CR1) and their control vector (sgLacZ) were purchased from Heyuan Biotechnology (Shanghai) Co., Ltd.

[0073] 5. In situ injection of pancreas C57BL / 6 mice (i.e. WT mice) were anesthetized and then injected into the pancreas. After deep anesthesia, the mice were injected intraperitoneally with sodium pentobarbital (50 mg / kg), and the skin and retroperitoneum were incised along the midline of the abdomen (incision length 0.5 cm) to expose the pancreas-spleen junction.

[0074] Aβ injection: Using an insulin syringe, 25 μL of Aβ oligomer solution (Aβ oligomer concentration of 100 μM, dissolved in PBS) or control solvent (PBS) was slowly injected at three points (head / body / tail) along the long axis of the pancreas. The peritoneum and skin were sutured layer by layer with absorbable sutures. After surgery, the mice were placed in a constant temperature resuscitation chamber and observed until they fully recovered spontaneous activity.

[0075] Viral vector injection: Using an insulin syringe, the CX3CR1 gene knockout lentiviral vector (sgCX3CR1) or control viral vector (sgLacZ) was slowly injected at three points (head / body / tail) along the long axis of the pancreas. The peritoneum and skin were sutured layer by layer with absorbable sutures. After surgery, mice were placed in a constant temperature resuscitation chamber and observed until they fully recovered spontaneous activity. Injection dosage: 2 μL of the original viral solution per mouse, or 50 μL of the viral solution diluted with PBS per mouse.

[0076] 6. Insulin and blood glucose testing After fasting for 2 hours, blood was collected from the tail vein of mice between 14:00 and 15:00, and blood glucose levels were measured immediately using a portable blood glucose meter and blood glucose test strips (from Bayer).

[0077] Microvenous blood was collected into heparin sodium anticoagulant tubes, centrifuged at 5000×g for 5 minutes at 4°C, and plasma was aliquoted and stored at -80°C. The absorbance was measured using a high-sensitivity mouse insulin ELISA kit (Mercodia, catalog number: 10-1049-01) and read by an ELISA reader.

[0078] 7. Immunohistochemistry 1) Tissue processing and perfusion fixation: Mice were anesthetized with sodium pentobarbital (50 mg / kg, intraperitoneal injection), and their hearts were perfused successively with pre-cooled PBS and 4% (w / v) PFA. Brain and pancreatic tissues were fixed in 4% (v / v) PFA for 24 hours, and then dehydrated in 30% (v / v) sucrose solution for 48 hours.

[0079] 2) Section preparation: Coronal sections of brain tissue (30 μm) and pancreatic sections (18 μm) were prepared using a cryostat. The brain sections were stored in an antifreeze solution (30% glycerol (v / v) + 30% (v / v) ethylene glycol / PBS) at -20°C.

[0080] 3) Staining and Quantification: Sections were rinsed three times with 0.1 M PBS buffer for 10 minutes each time. After rinsing, the sections were immersed in 5% (v / v) goat serum solution and blocked at room temperature for 2 hours. After blocking, the sections were picked out and placed in primary antibody dilution buffer, and incubated overnight at 4°C. The next day, the sections were picked out and rinsed three times with PBS buffer. Then, the sections were placed in fluorescent secondary antibody working solution and incubated at room temperature for 2 hours. After incubation, the sections were rinsed three times with PBS. Then, the sections were placed in DAPI dilution buffer, stained for 10 minutes, and rinsed three times with PBS buffer. After the final rinse, the sections were mounted on adhesive slides and allowed to dry before mounting with mounting medium. Finally, fluorescence images were acquired using a slide scanner or laser confocal microscopy. Cells were counted and fluorescence intensity values ​​were obtained using ImageJ software.

[0081] 4) Pancreatic Aβ detection: Pancreatic sections (18 μm) were co-stained with anti-Aβ antibody (brand: Abcam, catalog number: ab201060, dilution ratio: 1:500, labeled Aβ) and anti-insulin antibody (brand: CST, catalog number: 8138, dilution ratio: 1:1000, labeled islets) and islet-related Aβ deposition was analyzed by Imaris 9.9 three-dimensional reconstruction.

[0082] 5) Detection of CX3CR1 expression in pancreas: Pancreatic sections (18 μm) were co-stained with anti-CX3CR1 antibody (brand: Invitrogen, catalog number: PA1-28839; dilution ratio: 1:1000, labeled CX3CR1) and anti-insulin antibody (brand: CST, catalog number: 8138; dilution ratio: 1:1000, labeled islets) and DAPI. The knockout effect of CX3CR1 in pancreatic islets was analyzed by Imaris 9.9 three-dimensional reconstruction.

[0083] 6) Brain Aβ plaque analysis: Brain sections (30 μm) were blocked for 1.5 hours with blocking solution (5% (v / v) goat serum + 1% (v / v) BSA + 0.1% (v / v) Triton X-100 / TBS), and incubated at 4°C for 48 hours with anti-Aβ primary antibody (brand: Abcam, catalog number: ab134022, dilution ratio: 1:500, labeled with Aβ plaques). Images were acquired using a 10× objective lens (NA 0.45), and the area percentage of Aβ plaques in the hippocampus was calculated using ImageJ 1.53t.

[0084] 7) Brain GLUT1 detection: Brain sections (30 μm) were co-stained with anti-GLUT1 antibody (brand: Sigma, catalog number: MABS132, dilution ratio: 1:500, labeled glucose transporter 1); anti-CD31 antibody (brand: Sigma, catalog number: MAB1398z, dilution ratio: 1:500, labeled endothelial cells, used to identify vascular structures). 3-4 sections were taken from each mouse, and 3 fields of view were selected from each section to count glial cell density (cells / mm²).

[0085] 8) Assessment of glial cell proliferation: Brain sections (30 μm) were co-stained with anti-Iba1 antibody (brand: ABclonal, catalog number: A19776, dilution ratio: 1:500, for labeling microglia); anti-GFAP antibody (brand: Abcam, catalog number: ab7260, dilution ratio: 1:500, for labeling astrocytes). 3-4 sections were taken from each mouse, and 3 fields of view were selected from the radial layer of the hippocampus of each section to count glial cell density (cells / mm²).

[0086] 8. Protein electrophoresis and transfer (1) Sample processing for analysis of Akt signaling pathway: hCMEC / D3 cells (human brain microvascular endothelial cell line, from Zhejiang Meisen Cell Technology Co., Ltd.) were treated with human insulin for 6 hours and the expression of HIF1α, VEGF and GLUT1 proteins was measured.

[0087] (2) Protein lysis and extraction: Cells were lysed with pre-cooled RIPA lysis buffer (brand: Millipore, catalog number: 20-188). The lysis buffer contained phosphatase inhibitor (brand: Roche, catalog number: 4906837001) and protease inhibitor (brand: Roche, catalog number: 4693159001). The lysis buffer was centrifuged at 14,000 × g for 10 minutes at 4°C, and the supernatant was aliquoted and stored at -80°C.

[0088] (3) Protein concentration determination: The BCA method (Pierce) was used. TM The BCA protein assay kit (brand: ThermoScientific, catalog number: 23227) measures protein concentration.

[0089] (4) SDS-PAGE: 50 μg of protein was separated by electrophoresis on a 10% (v / v) SDS-polyacrylamide gel (brand: Bio-Rad, catalog number: 4561033) and transferred to a 0.2 μm nitrocellulose membrane (brand: GE Healthcare, catalog number 10600002) using the Bio-Rad Trans-Blot Turbo semi-dry transfer system (25 V, 30 minutes).

[0090] (5) Membrane blocking: 5% skim milk powder (brand: BD Difco, product number: 232100) was blocked in TBST solution (20 mM Tris, 150 mM NaCl, 0.1% (v / v) Tween-20, pH 7.6) at room temperature for 1 hour.

[0091] (6) Incubate with primary antibody at 4°C overnight: The sources and dosages of the antibodies are as follows: Anti-Akt2 antibody (brand: CST, catalog number: 3063; dilution ratio: 1:500); anti-p-Akt2 antibody (brand: CST, catalog number: 8599; dilution ratio: 1:500); anti-FOXO3 antibody (brand: CST, catalog number: 12829; dilution ratio: 1:500); anti-HIF1α antibody (brand: CST, catalog number: 36169; 1:500); anti-VEGF antibody (brand: Santa Cruz, catalog number: sc-53462; dilution ratio: 1:200); anti-GLUT1 antibody (brand: Millipore, catalog number: MABS132; dilution ratio: 1:500); anti-GAPDH antibody (brand: Sigma, catalog number: A5441; dilution ratio: 1:5000).

[0092] (7) Incubate the secondary antibody at room temperature for 2 hours The sources and dosages of the antibodies are as follows: HRP-labeled goat anti-rabbit IgG antibody (brand: Invitrogen, catalog number: 32260; dilution ratio: 1:1000); HRP-labeled goat anti-mouse IgG antibody (brand: Invitrogen, catalog number: 32230; dilution ratio: 1:1000).

[0093] (8) Detection and quantification: Development was performed using a SuperSignal™ West Pico PLUS chemiluminescent substrate (brand: Thermo Fisher, item number 34580), and exposure was recorded using a Tianneng chemiluminescence imager (brand: Tianneng, model: 5200). ImageJ 1.53 software (NIH) was used to quantify the grayscale values ​​of the bands, and GAPDH was used as an internal reference for calibration.

[0094] 9. New Object Recognition Experiment Experimental setup: The experiment used a white polypropylene open field apparatus (40×40×40 cm³), with two identical cylinders (5 cm in diameter) placed symmetrically 10 cm from the box wall.

[0095] Behavioral protocol: 1) Training phase: The mouse is placed facing the box wall and allowed to explore the object freely for 5 minutes. 2) Testing phase: After 1 hour, one of the objects is replaced with a novel cube (5 cm on each side). The sniffing time (nose tip ≤ 2 cm from the object) is recorded using ANY-maze 7.1 software.

[0096] Data Analysis: Discrimination Index = (Exploration Time of New Objects - Exploration Time of Familiar Objects) / Total Exploration Time × 100% 10. Water Maze Experiment Experimental setup: A circular pool (100 cm in diameter) is filled with 25°C clean water and mixed with non-toxic white pigment. A transparent acrylic platform (12 cm in diameter) is fixed in the target quadrant and submerged 1 cm below the water surface.

[0097] Training plan: Five consecutive days of training, four times a day (30-minute intervals). Each session lasts a maximum of 60 seconds. Those who cannot find the platform will be guided to the platform by the experimenter and stay there for 20 seconds.

[0098] Space exploration experiment: On day 6, the platform was removed, and the mice swam freely for 60 seconds. Ethovision XT 15.0 software was used to analyze the time spent in the target quadrant and the number of times the platform was crossed.

[0099] 11. 18 F-FDG-PET (18F-fluorodeoxyglucose positron emission tomography) imaging (1) Imaging protocol: 6-month-old and 8-month-old mice were injected with 13.5 MBq ¹ via the tail vein. 8 F-fluorodeoxyglucose (¹) 8 F-FDG). After 30 minutes of tracer ingestion while awake, the animal was anesthetized with a 2% isoflurane / oxygen mixture and underwent a 20-minute small animal PET scan. Respiratory rate (80-120 breaths / min) and body temperature (37.0±0.5°C) were monitored throughout the process.

[0100] (2) Image reconstruction and analysis: PET data reconstruction parameters: matrix: 136×131×315; voxel size: 0.38×0.38×0.54 mm³; Gaussian filter: half width at half maximum 1 mm; the hippocampus and cortical regions of interest (ROI) were selected using Amide 1.0.4-1 software, and the standardized uptake value (SUV) was calculated.

[0101] 12. Power Doppler angiography Imaging Method: A PodaMed-fUSI U10 functional brain ultrasound imaging system (Puda Medical Technology, Shanghai) was used, employing an L22-14 linear array probe (center frequency 18 MHz, 128 elements, element spacing 0.1 mm) for complete skull scanning. Ultrafast imaging parameters: 15-angle plane wave emission (-18° to +18°, step size 2.4°), pulse repetition frequency 7500 Hz, deflection interval 0.4 seconds, composite frame rate 500 Hz. 200 frames of radio frequency data were continuously acquired, and power Doppler signals were extracted using a singular value decomposition (SVD) clutter filtering algorithm.

[0102] 13. Tissue transparency, 3D fluorescence imaging, and quantitative analysis of the whole brain vascular network. (1) Organizational processing: Vascular markers: Mice were anesthetized with tribromoethanol, and their hearts were perfused with 80 μL of Lectin-649 vascular marker (tomato lectin-Alexa Fluor 649 conjugate, brand: Novogene, catalog number: NH-240525-DL649-1ml). After 10 minutes, the hearts were perfused with 20 mL of pre-cooled PBS and 20 mL of 4% (w / v) PFA.

[0103] Fixation and clearing: Brain tissue was fixed with 4% (w / v) PFA for 24 hours (with shaking at 50 rpm), and washed twice with PBS (2 hours each time). The whole brain was then soaked in the Nohhai Tissue Clearing Kit (brand: Nohhai Biotechnology, catalog number: NH-CR-230701) for 7 days to achieve whole-brain clearing.

[0104] (2) 3D imaging: The transparent whole brain was imaged using a Nohhai LS 18 flat-panel microscope with the following parameters: Excitation laser: 637 nm (100 mW); Objective lens: Olympus MVPLAPO 1× / 0.25NA; Z-axis step: 5 μm; Tiled scan: 4×4 grid, 10% overlap; The raw data were integrated using LS 18 image stitching software (v2.3.1), and the vascular network was reconstructed and analyzed using Amira 6.7.

[0105] (3) Quantitative analysis of the whole brain vascular network: 3D vascular reconstruction: Transparent whole-brain images were reconstructed in 3D using Imaris 3D software (v10.1.1, Oxford Instruments). The original data was resampled to isotropic voxels (500×500×500 μm³). A semi-automatic filament tracking and segmentation algorithm was used to extract vascular structures. The calculation formula is as follows: Blood vessel length density = Σ filament segment length (μm) / voxel volume (μm³) Fluorescence intensity density = total fluorescence intensity / blood vessel volume.

[0106] 14. Liquid-phase microarray detection of plasma inflammatory factors After the water maze behavioral experiment, blood was collected from the eyes of mice. After standing at room temperature for 1 hour, the blood was centrifuged at 5000×g for 5 minutes at 4°C, aliquoted, and stored at -80°C. The levels of serum cytokines including GM-CSF, IL-1α, IL-1β, IL-2, IL-6, IL-10, IL-13, and TNF-α were detected using the MILLIPLEX® Multiplex Protein Detection Kit, based on the protein multifactor liquid chromatography chip detection platform, commissioned to Mingyan Biotechnology (Shenzhen) Co., Ltd.

[0107] 15. Statistical Analysis Data are expressed as mean ± standard error and normality is verified by the Shapiro-Wilk test.

[0108] Two-group comparison: Two-tailed unpaired t-test Multiple group comparisons: one-way ANOVA + Tukey post-hoc test Significance markers: p<0.05, p<0.01, p<0.001 (GraphPad Prism 9.4.1).

[0109] Example 1: The damaging effect of Aβ on pancreatic insulin secretion Grouping of Aβ-induced AD model mice: At 3 months of age, C57BL / 6 mice were injected orally into the pancreas with Aβ oligomer solution and control solvent (PBS) (injection method is described in point 5 of the experimental method for orthotopic pancreatic injection). Aβ-induced AD model group (Aβ-O group, i.e., injected with Aβ oligomer) and normal control group (Vehicle group, i.e., injected with PBS) were prepared. Three months later, 6-month-old mice were taken from the tail vein to measure whole blood glucose and plasma insulin levels. After anesthesia, pancreatic tissue was taken to analyze pancreatic islet Aβ deposition. The effect of Aβ on pancreatic insulin secretion was preliminarily analyzed.

[0110] Grouping of 5xFAD transgenic AD model mice: Four-month-old normal control group (WT group) and 5xFAD transgenic AD model group (5xFAD group) mice were anesthetized and pancreatic tissue was collected to analyze pancreatic islet Aβ deposition. Three-month-old and eight-month-old normal control group (WT group) and 5xFAD transgenic AD model group (5xFAD group) mice were collected, and whole blood glucose and plasma insulin levels were measured by tail vein blood to further verify the damaging effect of Aβ on pancreatic insulin secretion.

[0111] Alzheimer's disease patient cohort grouping: Venous blood was collected from Alzheimer's disease patients (AD group) and age-matched controls (Non-AD group) to measure plasma insulin levels, further verifying the damaging effect of Aβ on pancreatic insulin secretion. The experimental groups are detailed in Table 1. In Table 1, the Healthy Control group consisted of age-matched and AD-matched healthy individuals without mental illness, while the Non-AD group consisted of age-matched and AD-matched patients without Alzheimer's disease but with other neuropsychiatric disorders.

[0112] Test results as follows Figures 1-3 .

[0113] Figure 1 The damaging effects of Aβ on pancreatic insulin secretion (in Aβ-induced AD model mice): Figure 1 (a) is a schematic diagram of an animal experiment; Figure 1 (bc) represents pancreatic Aβ deposition analysis (immunofluorescence staining); Figure 1 (d) shows the effect of pancreatic Aβ injection on plasma insulin levels in mice; Figure 1 (e) shows the effect of pancreatic Aβ injection on whole blood glucose levels in mice; Figure 2 The damaging effects of Aβ on pancreatic insulin secretion (in 5xFAD transgenic AD model mice): Figure 2 The middle section (ab) represents pancreatic Aβ deposition analysis (immunofluorescence staining). Figure 2 (c) is a schematic diagram of the blood collection experiment in 5xFAD mice; Figure 2 The value in (de) represents the mouse plasma insulin level (3M, 8M). Figure 2 (fg) represents the whole blood glucose level in mice (3M, 8M); Figure 3 The impaired effect of Aβ on pancreatic insulin secretion (in AD patients).

[0114] Aberrant Aβ deposition in the brain is one of the main pathological features of Alzheimer's disease (AD) and a significant driving factor in its development and progression. Aβ deposition is not only reported in the cerebral cortex of AD patients, but also in peripheral tissues such as the pancreas. Insulin plays a crucial role not only in regulating blood glucose levels but also in the development and progression of AD. Studies have found that insulin deficiency impairs brain metabolism, but the exact function of insulin in the brain is not fully understood.

[0115] This study used C57BL / 6 mice to investigate the effect of Aβ on pancreatic function. Aβ-induced AD model mice (Aβ-O group) were established by orthotopic injection of Aβ oligomers into the pancreas of 3-month-old C57BL / 6 mice. Control mice (Vehicle group) received orthotopic injection of PBS (PBS) into their pancreas. Three months later, whole blood glucose and plasma insulin levels were measured in 6-month-old mice, and immunofluorescence staining was used to observe Aβ deposition in the pancreatic islets. The experimental results are as follows: Figure 1 As shown, compared with the control group mice (Vehicle group), 6-month-old Aβ-induced AD model mice (Aβ-O group) showed a significant increase in Aβ accumulation in the pancreatic islets and a significant decrease in plasma insulin levels, but no significant change in whole blood glucose levels, which preliminarily demonstrates the damaging effect of Aβ on pancreatic insulin secretion function.

[0116] In this embodiment, to verify the effect of Aβ on pancreatic function, immunofluorescence staining was used to observe the pancreatic islet Aβ deposition in 4-month-old 5xFAD transgenic AD model mice (5xFAD group) and isotype control mice (WT group). Whole blood glucose and plasma insulin levels were also measured in 3-month-old and 8-month-old 5xFAD transgenic AD model mice and isotype control mice. The experimental results are as follows: Figure 2 As shown, compared with the age-matched control group (WT group), the accumulation of Aβ in the pancreatic islets of 4-month-old 5xFAD transgenic AD model mice (5xFAD group) was significantly increased, and the plasma insulin level of 3-month-old and 8-month-old 5xFAD transgenic AD model mice (5xFAD group) was significantly reduced, but the whole blood glucose level did not change significantly, which verified the damaging effect of Aβ on pancreatic insulin secretion function.

[0117] Table 1. Alzheimer's patient cohort

[0118] To further verify the effect of Aβ on pancreatic function, this embodiment collected plasma insulin level data from Alzheimer's disease patients (AD group) and age-matched controls (Non-AD group). The results are as follows: Figure 3 As shown, compared with the age-matched control group (Non-AD group), the incidence of Alzheimer's disease (AD group) was significantly reduced, further verifying the damaging effect of Aβ on pancreatic insulin secretion.

[0119] Example 2: The damaging effect of Aβ on pancreatic secretion in mice is dependent on CX3CR1. To investigate the role of peripheral pancreatic CX3CR1 in the Aβ-induced impaired pancreatic secretion in mice, this study conducted the following two experiments.

[0120] (1) Injection of lentivirus into the pancreas (the injection method is described in point 5 of the experimental method for viral vector injection): Specific knockout of mouse pancreatic CX3CR1 gene experiment: 3-month-old C57BL / 6 mice were injected orally into the pancreas with CX3CR1 gene knockout lentivirus vector (sgCX3CR1) and control viral vector (sgLacZ). After 3 months, the mice were anesthetized and the pancreatic tissue was fixed and sectioned. The expression of pancreatic CX3CR1 was analyzed by immunohistochemistry to verify the knockout effect of lentivirus on mouse pancreatic CX3CR1. At the same time, the plasma insulin level was measured by taking tail vein blood from 6-month-old mice.

[0121] (2) Establishment of a mouse experiment with specific knockout of pancreatic CX3CR1 gene based on the Cre-LoxP system: First, the Cre-LoxP recombination system, Pdx Cre Transgenic mice (pancreas-specific tool mice) and CX3CR1 loxp / loxp Transgenic mouse hybridization to construct Pdx that specifically knocks out CX3CR1 in pancreatic tissue. Cre CX3CR1 loxp / loxp Transgenic mice. 5xFAD transgenic mice and CX3CR1 loxp / loxp Transgenic mouse hybridization to construct CX3CR1 loxp / loxp 5xFAD transgenic mice. Pdx Cre Transgenic mice and CX3CR1 loxp / loxp ; 5xFAD transgenic mice were crossed, and CX3CR1-specific knockout mice, namely Pdx, were constructed in the context of 5xFAD transgenic AD model mice. Cre CX3CR1 loxp / loxp 5xFAD transgenic mice. Then, 6-month-old Pdx mice were used. Cre CX3CR1 loxp / loxp Transgenic mice and age-matched wild-type mice (WT group) were anesthetized, and pancreatic tissue sections were fixed. Immunohistochemistry was used to analyze the expression of CX3CR1 in the pancreas, verifying the knockout effect of the Cre-LoxP system on CX3CR1 in the mouse pancreas. Finally, 6-month-old Pdx mice were... Cre CX3CR1 loxp / loxp ; 5xFAD transgenic mice, Pdx Cre CX3CR1 loxp / loxp Blood samples were taken from the tail vein of transgenic mice and age-matched wild-type mice (WT group) to measure plasma insulin levels.

[0122] Test results as follows Figures 4-5 As shown.

[0123] Figure 4The impaired effect of Aβ on pancreatic secretion is dependent on CX3CR1 (experimental study of silencing CX3CR1 expression in the pancreas by in situ injection of LV viral vector): Figure 4 (a) is a schematic diagram of the experiment to silence CX3CR1 expression in the pancreas by in situ injection of LV virus vector; Figure 4 (bc) In the middle, the LV virus was injected into the pancreas of mice to silence the expression of CX3CR1 in the pancreas. Immunofluorescence staining was performed 3 months later to verify the silencing effect on pancreatic CX3CR1. Figure 4 Effect of (d) on plasma insulin levels in mice; Figure 5 The impaired pancreatic secretion effect of Aβ is dependent on CX3CR1 (conditional knockout of CX3CR1 in transgenic mice): Figure 5 (a) is a schematic diagram of the conditional knockout of CX3CR1 in transgenic mice. Figure 5 (b) shows the effect of CX3CR1 knockout in the pancreas verified by immunofluorescence staining. Figure 5 (c) shows the effect on plasma insulin levels in mice.

[0124] The CX3CL1-CX3CR1 axis plays a vital role in an increasing number of studies. In the development and progression of Alzheimer's disease (AD), CX3CR1 is an important microglial immune checkpoint. CX3CL1 and CX3CR1 are expressed in spinal cord neurons and microglia, respectively, and the CX3CR1-CX3CL1 signaling plays a crucial role in maintaining microglial function and regulating interactions between neurons and astrocytes. Studies have found that CX3CR1 deficiency impairs microglial cell turnover in the brain, exacerbating AD symptoms in 5xFAD mice, including Aβ-induced Tau protein phosphorylation, neurodegeneration, synaptic dysfunction, and cognitive decline.

[0125] In this embodiment, to investigate the effect of CX3CR1 on the impaired secretory function of pancreatic islets caused by Aβ, 3-month-old mice were anesthetized and injected into the pancreas with either a lentiviral vector (sgCX3CR1) or a control vector (sgLacZ). The experimental results are as follows: Figure 4As shown, immunohistochemistry revealed a decrease of approximately 50% in CX3CR1 expression in the pancreas of 6-month-old mice (sgCX3CR1 group), validating the knockout effect of pancreatic lentiviral sgCX3CR1 vector injection on pancreatic CX3CR1. Compared with age-matched control mice (WT_sgLacZ group), plasma insulin levels were significantly lower in 6-month-old 5xFAD transgenic AD model mice (5xFAD_sgLacZ group); compared with age-matched 5xFAD transgenic AD model mice (5xFAD_sgLacZ group), plasma insulin levels were significantly higher in 5xFAD transgenic AD model mice (5xFAD_sgLacZ group) 3 months after orthotopic injection of lentiviral sgCX3CR1 vector into the pancreas, indicating that the damaging effect of Aβ on pancreatic islet secretory function is dependent on CX3CR1.

[0126] In this embodiment, to further investigate the role of CX3CR1 in Aβ-induced pancreatic islet secretory function, a pancreas-specific CX3CR1 knockout mouse model was established using the Cre-LoxP recombination system. Experimental results are as follows: Figure 5 As shown, compared with the age-matched control group (WT group), 6-month-old Pdx was observed by immunohistochemistry. Cre CX3CR1 loxp / loxp Transgenic mice showed significantly reduced expression of CX3CR1 protein in the pancreas, validating the knockout effect of CX3CR1 in the mouse pancreas. Compared with age-matched control mice (WT group), 6-month-old 5xFAD transgenic AD model mice (CX3CR1) showed significantly reduced expression. loxp / loxp Plasma insulin levels were significantly reduced in the 5xFAD group; compared with age-matched 5xFAD transgenic AD model mice (CX3CR1) loxp / loxp Compared to the 5xFAD group, the constructed pancreas-specific knockout CX3CR1 5xFAD mice (Pdx) Cre CX3CR1 loxp / loxp The plasma insulin level in the 5xFAD group was significantly elevated, almost returning to the level of the age-matched control group (WT group), further demonstrating that the damaging effect of Aβ on pancreatic islet secretory function depends on CX3CR1.

[0127] Example 3: Therapeutic effect of insulin on Aβ-induced AD model mice induced by in situ injection of insulin into pancreatic islets Aβ-induced AD model mouse experiment: Three-month-old C57BL / 6 mice were orally injected with Aβ oligomer solution and control solvent (PBS) to prepare normal control group (Veh group) and Aβ-induced AD model group (Aβ-O-Veh group) mice, respectively. Three months later, some normal control group (Veh group) and Aβ-induced AD model group (Aβ-O-Veh group) mice were treated by subcutaneous injection of insulin (i.e., human insulin injection solution, from Eli Lilly Suzhou Pharmaceutical Co., Ltd., concentration of 100U / mL, diluted with physiological saline to ensure consistent injection volume) (1U / kg, once a day, for 10 consecutive days), which became insulin treatment control group (Veh-Insulin group) and insulin treatment Aβ-induced AD model group (Aβ-O-Insulin group) mice. The remaining normal control group (Veh group) and Aβ-induced AD model group (Aβ-O-Veh group) mice were injected with the same volume of solvent (physiological saline), with an injection volume of 0.1 ml / 20g / day.

[0128] At the end of the experiment, blood was collected from the tail vein of mice to measure whole blood glucose and plasma insulin levels. Cognitive function of the mice was assessed using a novel object recognition experiment and a water maze behavioral experiment. 18 F-FDG small animal PET imaging was used to detect brain glucose metabolism, and immunohistochemistry was used to detect the expression of GLUT1 / CD31 in brain tissue.

[0129] Test results as follows Figures 6-9 As shown.

[0130] Figure 6 Effects of exogenous insulin intake on plasma insulin and glucose levels in AD model mice (in Aβ-induced AD model mice): Figure 6 (a) is a schematic diagram of an animal experiment; Figure 6 (b) shows the plasma insulin level in mice; Figure 6 (c) represents the whole blood glucose level in mice; Figure 7 The effect of exogenous insulin intake on cognitive impairment in AD model mice (in Aβ-induced AD model mice): Figure 7 (a) shows the new object recognition experiment; Figure 7 (bc) Water maze experiment; Figure 8 The effect of exogenous insulin intake on brain glucose metabolism in AD model mice (in Aβ-induced AD model mice). Figure 9 The effects of exogenous insulin intake on vascular structure and function in AD model mice (in Aβ-induced AD model mice).

[0131] This embodiment evaluated the effect of insulin intervention on peripheral glucose and insulin levels in Aβ-induced AD model mice by measuring whole blood glucose and plasma insulin levels. The results are as follows: Figure 6 As shown, compared with the normal control group (Veh group), the plasma insulin level of mice in the Aβ-induced AD model group (Aβ-O-Veh group) was significantly reduced, while the whole blood glucose level did not change significantly. In the Aβ-induced AD model group, after insulin intervention, the plasma insulin level of mice in the Aβ-O-Insulin group significantly increased, almost returning to the plasma insulin level of the normal control group, while the whole blood glucose level did not change significantly. In the normal control group (Veh group), after insulin intervention, the plasma insulin and whole blood glucose levels of mice in the Veh-Insulin group did not change significantly. These results indicate that exogenous insulin supplementation can restore plasma insulin levels in mice in the Aβ-induced AD model group.

[0132] This embodiment evaluated the effects of insulin intervention on cognitive function in Aβ-induced AD model mice through novel object recognition and water maze tests. The novel object recognition test, which utilizes an animal's tendency to explore novel objects to assess its memory and object recognition abilities, has become a standard tool for evaluating animal memory abilities under various physiological and pathological conditions. Results are as follows... Figure 7 As shown, compared with the normal control group (Veh group), mice in the Aβ-induced AD model group (Aβ-O-Veh group) spent less time exploring new objects, while mice in the Aβ-induced AD model group (Aβ-O-Insulin group) showed a significant increase in exploration time for new objects after insulin intervention. The water maze is a classic method for assessing learning and memory in animal models and is widely used in research on spatial learning and memory abilities in rodents, as well as cognitive impairment and aging. Results are as follows... Figure 7 As shown, during the water maze training phase, compared with the normal control group (Veh group), the Aβ-induced AD model group (Aβ-O-Veh group) mice took longer to reach the platform, while the Aβ-induced AD model mice (Aβ-O-Insulin group) took shorter times to reach the platform after insulin intervention. During the water maze test phase, compared with the normal control group (Veh group), the Aβ-induced AD model group (Aβ-O-Veh group) mice spent less time in the target quadrant, while the Aβ-induced AD model mice (Aβ-O-Insulin group) spent significantly more time in the target quadrant after insulin intervention. These results indicate that exogenous insulin supplementation can improve cognitive and memory impairment in Aβ-induced AD model mice.

[0133] This embodiment involves treating mice. 18 F-FDG–PET scanning was used to investigate the effect of insulin intervention on glucose utilization in the brains of mice with Aβ-induced AD model.18 F-FDG-PET is an important tool for the diagnosis, assessment of progression, and prognosis of Alzheimer's disease. 18 F-FDG ( 18 F-fluorodeoxyglucose is a fluorinated derivative of 2-deoxyglucose. 18 F-FDG-PET can assess the degree and location of hypometabolism, reflecting neuronal dysfunction. In dementia patients, it is used... 18 Low brain metabolism detected by F-FDG-PET is a marker of neurodegeneration. 18 F-FDG-PET measurements were performed on regional glucose consumption that was directly correlated with the local intensity of brain glutamate synapses and astrocyte activity. Results were as follows: Figure 8 As shown, compared with the normal control group (Veh group), the brains of mice in the Aβ-induced AD model group (Aβ-O-Veh group) showed lower levels of... 18 F-FDG signaling, brain of Aβ-induced AD model mice (Aβ-O-Insulin group) after insulin intervention 18 Enhanced F-FDG signaling indicates that supplementing with exogenous insulin can increase glucose utilization in the brains of Aβ-induced AD model mice.

[0134] This embodiment uses immunofluorescence staining to observe the effects of insulin intervention on angiogenesis and glucose transport capacity in the brain of Aβ-induced AD model mice. Glucose transporter 1 (GLUT1) is the most important transport protein for transporting glucose from the blood to the brain parenchyma. GLUT1 is mainly expressed in vascular endothelial cells and the terminal foot of astrocytes, and is crucial for maintaining the integrity of the brain capillary network and the blood-brain barrier. CD31 is a platelet endothelial cell adhesion molecule, mainly used to demonstrate the presence of endothelial cell tissue and assess angiogenesis. Immunofluorescence staining results are shown below. Figure 9 As shown, compared with the normal control group (Veh group), there was no significant change in CD31 expression in the brain of Aβ-induced AD model mice (Aβ-O-Veh group), but the proportion of GLUT1 green fluorescence signal to CD31 red fluorescence signal was significantly reduced. After insulin intervention, the proportion of GLUT1 green fluorescence signal to CD31 red fluorescence signal in the brain of Aβ-induced AD model mice (Aβ-O-Insulin group) was significantly enhanced, indicating that supplementing with exogenous insulin can increase the glucose uptake and utilization capacity of Aβ-induced AD model mice.

[0135] Example 4: Therapeutic effect of insulin on 5xFAD transgenic AD model mice Experiment on 5xFAD transgenic AD model mice: Some 6-month-old normal control group (WT-Veh group) and 5xFAD transgenic AD model group (5xFAD-Veh group) mice were treated by subcutaneous injection of insulin (i.e., human insulin injection solution, from Eli Lilly Suzhou Pharmaceutical Co., Ltd., concentration of 100U / mL, diluted with physiological saline to the injection concentration before use to ensure consistent injection volume) (1U / kg, once / day, for 10 consecutive days). These were the insulin treatment group (WT-Insulin group) and the insulin-treated 5xFAD transgenic AD model group (5xFAD-Insulin group) mice. The remaining normal control group (WT-Veh group) and 5xFAD transgenic AD model group (5xFAD-Veh group) mice were injected with the same volume of solvent (physiological saline), with an injection volume of 0.1 ml / 20g / day.

[0136] At the end of the experiment, blood was collected from the tail vein of mice to measure whole blood glucose and plasma insulin levels. Cognitive function of the mice was assessed using a novel object recognition experiment and a water maze behavioral experiment. 18 F-FDG small animal PET imaging was used to detect brain glucose metabolism, immunohistochemistry was used to detect the expression of GLUT1 / CD31, Aβ, GFAP, and Iba1 in brain tissue, color Doppler blood flow imaging was used to detect cerebral blood flow, transparent brain technology 3D fluorescence imaging was used to quantitatively analyze the whole brain vascular network, and liquid phase chip was used to detect the level of inflammatory factors in mouse plasma.

[0137] Test results as follows Figures 10-19 As shown.

[0138] Figure 10 The effect of exogenous insulin intake on plasma insulin and glucose levels in AD model mice (in 5xFAD transgenic AD model mice). Figure 11 The effect of exogenous insulin intake on cognitive impairment in AD model mice (in 5xFAD transgenic AD model mice). Figure 11 (a) shows the new object recognition experiment; Figure 11 (b) shows the water maze experiment; Figure 12 The effect of exogenous insulin intake on brain glucose metabolism in AD model mice (in 5xFAD transgenic AD model mice). Figure 13 The effects of exogenous insulin intake on vascular structure and function in AD model mice (in 5xFAD transgenic AD model mice). Figure 14 Effects of exogenous insulin intake on the whole brain microvascular system in AD model mice (3D fluorescence imaging of whole brain microvessels labeled with lectin) (in 5xFAD transgenic AD model mice); Figure 15 Effects of exogenous insulin intake on cerebral blood flow signals in AD model mice (color Doppler blood flow imaging) (in 5xFAD transgenic AD model mice); Figure 16 The effect of exogenous insulin intake on serum inflammatory factors in AD model mice (in 5xFAD transgenic AD model mice); Figure 17 Effects of exogenous insulin intake on astrocytes in the brain of AD model mice (in 5xFAD transgenic AD model mice). Figure 18 The effect of exogenous insulin intake on microglia in the brain of AD model mice (in 5xFAD transgenic AD model mice). Figure 19 The effect of exogenous insulin intake on brain Aβ deposition in AD model mice (in 5xFAD transgenic AD model mice).

[0139] This embodiment evaluated the effect of insulin intervention on peripheral glucose and insulin levels in 5xFAD transgenic AD model mice by measuring whole blood glucose and plasma insulin levels. Results are as follows: Figure 10 As shown, compared with the normal control group (WT-Veh group), the plasma insulin level of mice in the 5xFAD transgenic AD model group (5xFAD-Veh group) was significantly reduced, while the whole blood glucose level did not change significantly. After insulin intervention, the plasma insulin level of mice in the 5xFAD-Insulin group increased significantly, almost returning to the plasma insulin level of the normal control group, while the whole blood glucose level did not change significantly. These results indicate that exogenous insulin supplementation can restore plasma insulin levels in 5xFAD transgenic AD model mice.

[0140] This embodiment evaluated the effects of insulin intervention on cognitive function in 5xFAD transgenic AD model mice through novel object recognition and water maze tests. The results of the novel object recognition test are as follows: Figure 11 As shown, compared with the normal control group (WT-Veh group), mice in the 5xFAD transgenic AD model group (5xFAD-Veh group) spent less time exploring new objects, while mice in the 5xFAD-Insulin group (5xFAD-Insulin group) showed a significant increase in exploration time for new objects after insulin intervention. The results of the water maze experiment are as follows... Figure 11As shown, during the water maze training phase, compared with the normal control group (WT-Veh group), mice in the 5xFAD transgenic AD model group (5xFAD-Veh group) took longer to reach the platform, while mice in the 5xFAD-Insulin group (after insulin intervention) took shorter times to reach the platform. During the water maze test phase, compared with the normal control group (WT-Veh group), mice in the 5xFAD transgenic AD model group (5xFAD-Veh group) spent less time in the target quadrant, while mice in the 5xFAD-Insulin group (after insulin intervention) spent significantly more time in the target quadrant. These results indicate that exogenous insulin supplementation can improve cognitive and memory impairment in 5xFAD transgenic AD model mice.

[0141] This embodiment involves treating mice. 18 F-FDG–PET scan was used to investigate the effect of insulin intervention on glucose utilization in the brains of 5xFAD transgenic AD model mice. 18 F-FDG-PET is an important tool for the diagnosis, assessment of progression, and prognosis of Alzheimer's disease. 18 F-FDG-PET can assess the degree and location of hypometabolism, reflecting neuronal dysfunction. 18 F-FDG–PET scan results are as follows Figure 12 As shown, compared with the normal control group (WT-Veh group), the brains of mice in the 5xFAD transgenic AD model group (5xFAD-Veh group) showed lower levels of... 18 F-FDG signaling, brain of mice after insulin intervention (5xFAD-Insulin group) 18 Enhanced F-FDG signaling indicates that supplementation with exogenous insulin can increase glucose utilization in the brains of 5xFAD transgenic AD model mice.

[0142] This embodiment uses immunofluorescence staining to observe the effects of insulin intervention on cerebral angiogenesis and glucose transport capacity in 5xFAD transgenic AD model mice. The immunofluorescence staining results are as follows: Figure 13 As shown, compared with the normal control group (WT-Veh group), the expression of CD31 and the proportion of GLUT1 green fluorescence signal to CD31 red fluorescence signal in the brains of mice in the 5xFAD transgenic AD model group (5xFAD-Veh group) were significantly reduced. After insulin intervention, the expression of CD31 and the proportion of GLUT1 green fluorescence signal to CD31 red fluorescence signal in the brains of mice in the 5xFAD transgenic AD model group were significantly enhanced, indicating that supplementing with exogenous insulin can increase the uptake and utilization of glucose in the brains of 5xFAD transgenic AD model mice.

[0143] This embodiment utilizes tissue transparency technology and 3D imaging technology to reconstruct and visualize the microvessels in the whole brain of mice, observing the effect of insulin intervention on the microvascular network in the brain of 5xFAD transgenic AD model mice. Using lectins as staining dyes, capillary structures in mice can be rapidly labeled, clearly revealing blood vessels and their complex branching structures. Results are as follows... Figure 14 As shown, compared with the normal control group (WT-Veh group), the vascular length and vascular fluorescence intensity in the cerebral cortex and hippocampus of mice in the 5xFAD transgenic AD model group (5xFAD-Veh group) were significantly reduced. After insulin intervention, the vascular length and vascular fluorescence intensity in the cerebral cortex and hippocampus of mice in the 5xFAD-Insulin group were significantly increased, suggesting that exogenous insulin supplementation can restore the microvascular structure of the brain in 5xFAD transgenic AD model mice.

[0144] This embodiment uses a Doppler blood flow system to detect cerebral blood flow perfusion in mice and observes the effect of insulin intervention on cerebral blood flow in 5xFAD transgenic AD model mice. Changes in cerebral vascular structure and function may lead to an increased risk of impaired cerebral blood flow circulation and neurodegeneration. The results of color Doppler blood flow imaging are shown below. Figure 15 As shown, compared with the normal control group (WT-Veh group), the cerebral blood flow fluorescence intensity of mice in the 5xFAD transgenic AD model group (5xFAD-Veh group) was significantly reduced, while the cerebral blood flow fluorescence intensity of mice after insulin intervention (5xFAD-Insulin group) was significantly enhanced, indicating that supplementing with exogenous insulin can restore cerebral blood flow in 5xFAD transgenic AD model mice.

[0145] This embodiment observes the effect of insulin intervention on inflammation in 5xFAD transgenic AD model mice by measuring serum cytokine levels. Cytokines participate in the body's immune and inflammatory responses; detecting the levels of these cytokines can assess the body's immune status and inflammatory response. The results of liquid-phase microarray detection of inflammatory factors are shown below. Figure 16 As shown, compared with the normal control group (WT-Veh group), the serum levels of cytokines such as IL-13, GM-CSF, IL-2, IL-1β, and TNF-α in mice in the 5xFAD transgenic AD model group (5xFAD-Veh group) were significantly increased. After insulin intervention, the serum levels of cytokines such as IL-13, GM-CSF, IL-2, IL-1β, and TNF-α in mice (5xFAD-Insulin group) were decreased, and the levels of IL-1α and IL-10 cytokines were also decreased. This indicates that supplementing with exogenous insulin can improve the inflammatory response in 5xFAD transgenic AD model mice.

[0146] This embodiment uses immunofluorescence staining to observe the effects of insulin intervention on astrocytes, microglia, and Aβ deposition in the brains of 5xFAD transgenic AD model mice. The results of immunofluorescence staining of brain sections are shown below. Figure 17 , Figure 18 , Figure 19 As shown, compared with the 5xFAD transgenic AD model group (5xFAD-Veh group) mice, the fluorescence area of ​​GFAP+ astrocytes and Iba1+ microglia in brain slices of mice after insulin intervention (5xFAD-Insulin group) decreased significantly, while the fluorescence area of ​​Aβ+ cells did not change significantly. This suggests that supplementing with exogenous insulin can inhibit the excessive activation of astrocytes and microglia in the brains of 5xFAD transgenic AD model mice.

[0147] Example 5: Effects of insulin on the insulin signaling pathway in endothelial cells In in vitro experiments, hCMEC / D3 cells (human brain microvascular endothelial cell line, obtained from Zhejiang Meisen Cell Technology Co., Ltd.) were treated with human insulin. After 6 hours, the cells were collected to measure the expression of proteins such as FOXO3, pAKT2, AKT2, Hif1α, VEGF, and GLUT1. The supernatant of the culture medium was collected to measure the levels of Akt1, Akt3, FOXO4, and INSR secreted by hCMEC / D3 cells, in order to explore the role of insulin in microvascular cell signaling pathways.

[0148] The procedure for treating endothelial cells with human insulin was as follows: The insulin solution (concentration: 9.5-11.5 mg / mL, brand: Sigma-Aldrich, catalog number: I9278) was diluted with hCMEC / D3 complete culture medium to prepare working solutions of different concentrations (0 nM, 1 nM, 10 nM); 5 × 10 6 hCMEC / D3 cells were seeded into 12-well plates containing complete hCMEC / D3 culture medium at a rate of cells / well. After culturing in a cell culture incubator for 24 hours, the medium was replaced with the above-mentioned working solution and treated in a cell culture incubator for 6 hours. Each concentration of working solution was used to prepare three 12-well plates.

[0149] Western blotting was used to determine the expression of proteins such as FOXO3, pAKT2, AKT2, Hif1α, VEGF, and GLUT1 in cells (see section 8 of the experimental methods for detailed methods on protein electrophoresis and transfer).

[0150] Protein concentrations in culture medium supernatant were determined by ELISA: The concentrations of Akt1, Akt3, FOXO4, and INSR in the culture medium supernatant were determined using an ELISA kit (Insulin / IGF-1 SignalingPathway ELISA Sampler Pack, brand: AssayGenie, catalog number: SKFI0003).

[0151] Test results as follows Figures 20-21 As shown.

[0152] Figure 20 The figure shows the results of detecting the effects of insulin on the expression of FOXO3, pAKT2, AKT2, GLUT1, HIF1α and VEGF proteins in hCMEC / D3 cells; Figure 21 The figure shows the results of detecting the effects of insulin on the levels of Akt1, Akt3, FOXO4 and INSR secreted by hCMEC / D3 cells.

[0153] This embodiment uses insulin treatment on hCMEC / D3 cells to investigate the effect of insulin intervention on hCMEC / D3 cell signaling pathways. The human brain microvascular endothelial cell line (hCMEC / D3) is a commonly used human blood-brain barrier (BBB) ​​model, which can be used for neuroscience and neurodegenerative disease research. In vitro cell experiment results are as follows... Figure 20 , Figure 21 As shown, insulin induces increased expression of AKT2, pAKT2, Hif1α, VEGF, and GLUT1 proteins in hCMEC / D3 cells and inhibits FOXO3 protein expression, but has no significant effect on the expression of Akt1, Akt3, FOXO4, and INSR proteins secreted by hCMEC / D3 cells. This suggests that insulin regulates the insulin signaling pathway in human brain microvascular endothelial cells through AKT2 / FOXO3.

[0154] Example 6: Therapeutic effects of different doses of insulin on 5xFAD transgenic AD model mice Experiment on 5xFAD transgenic AD model mice: A subset of 6-month-old 5xFAD transgenic AD model mice (5xFAD-Veh group) were intervened by subcutaneous injection of different doses of insulin (i.e., human insulin injection solution, sourced from Eli Lilly Suzhou Pharmaceutical Co., Ltd., concentration 100 U / mL, diluted with physiological saline to the required injection concentration to ensure consistent injection volume) (0.25 U / kg, 0.5 U / kg, 1 U / kg, 2 U / kg, 4 U / kg, once daily for 10 consecutive days). These were designated as different insulin treatment groups (5xFAD-Insulin 0.25 group, 5xFAD-Insulin 0.5 group, 5xFAD-Insulin 1.0 group, 5xFAD-Insulin 2.0 group, 5xFAD-Insulin...). The remaining mice in the 5xFAD transgenic AD model group (5xFAD-Veh group) and the normal control group (WT-Veh group) were injected with the same volume of solvent (physiological saline), with an injection volume of 0.1 ml / 20g / day.

[0155] At the end of the experiment, blood was taken from the tail vein of mice to measure whole blood glucose and plasma insulin levels. The cognitive function of the mice was tested through a new object recognition experiment and a water maze behavioral experiment.

[0156] Test results as follows Figure 22 As shown, where Figure 22 (a) Schematic diagram of the experiment; Figure 22 (bc) shows the effect of different doses of insulin intake on whole blood glucose and plasma insulin levels in AD model mice (in 5xFAD transgenic AD model mice). Figure 22 (def) represents the effect of exogenous insulin intake on cognitive impairment in AD model mice (in 5xFAD transgenic AD model mice). Figure 22 (d) shows the new object recognition experiment. Figure 22 The middle (ef) represents the water maze experiment; This embodiment evaluated the effects of different doses of insulin intervention on peripheral glucose and insulin levels in 5xFAD transgenic AD model mice by measuring whole blood glucose and plasma insulin levels. Results are as follows: Figure 22As shown, compared with the normal control group (WT-Veh group), the plasma insulin level of mice in the 5xFAD transgenic AD model group (5xFAD-Veh group) was significantly reduced, while the whole blood glucose level did not change significantly. After insulin intervention (0.5 U / kg, 1.0 U / kg, 2.0 U / kg, 4.0 U / kg, once a day for 10 consecutive days), the plasma insulin level of 5xFAD transgenic AD mice increased significantly. The whole blood glucose level of 5xFAD transgenic AD mice in the 5xFAD-Insulin 0.5 group, 5xFAD-Insulin 1.0 group, and 5xFAD-Insulin 2.0 group did not change significantly, but the whole blood glucose level of 5xFAD transgenic AD mice in the 5xFAD-Insulin 4.0 group decreased, which may lead to hypoglycemia symptoms. The above results indicate that insulin supplementation can restore plasma insulin levels in 5xFAD transgenic AD model mice and exhibits a certain dose-dependent effect.

[0157] This embodiment evaluated the effects of insulin intervention on cognitive function in 5xFAD transgenic AD model mice through novel object recognition and water maze tests. The results of the novel object recognition test are as follows: Figure 22 As shown, compared with the normal control group (WT-Veh group), mice in the 5xFAD transgenic AD model group (5xFAD-Veh group) spent less time exploring new objects. After insulin intervention (0.5 U / kg, 1.0 U / kg, 2.0 U / kg, 4.0 U / kg, once daily for 10 consecutive days), the exploration time of mice in the 5xFAD-Insulin group significantly increased. The results of the water maze experiment are shown below. Figure 22 As shown, during the water maze training phase, compared with the normal control group (WT-Veh group), the mice in the 5xFAD transgenic AD model group (5xFAD-Veh group) took longer to reach the platform. After insulin intervention (0.5U / kg, 1U / kg, 2U / kg, 4U / kg, once a day for 10 consecutive days), the mice in the 5xFAD transgenic AD model group (5xFAD-Veh group) took shorter to reach the platform. During the water maze test phase, compared with the normal control group (WT-Veh group), the mice in the 5xFAD transgenic AD model group (5xFAD-Veh group) spent less time in the target quadrant. After insulin intervention (0.5U / kg, 1U / kg, 2U / kg, 4U / kg, once a day for 10 consecutive days), the mice in the 5xFAD transgenic group spent significantly more time in the target quadrant, showing a certain dose-dependent effect. The above experimental results show that supplementing with a certain dose of exogenous insulin (0.5U / kg, 1.0U / kg, 2.0 U / kg, once a day for 10 consecutive days) can significantly improve cognitive and memory impairment in 5xFAD transgenic AD model mice.

[0158] This application describes the formulation of insulin and its analogues as drugs for treating Alzheimer's disease. Insulin and its analogues compensate for the reduction in pancreatic insulin secretion caused by Aβ by supplementing the body's insulin levels. By taking in insulin from exogenous sources, they restore the body's insulin levels and microenvironment, thereby restoring plasma insulin levels in AD model mice. They have therapeutic effects such as improving cognitive dysfunction, improving cerebrovascular lesions, restoring brain glucose metabolism function, and reducing brain inflammation in AD model mice. This provides a definite, safe, and affordable alternative drug for the prevention and treatment of Alzheimer's disease, expands the indications for insulin and its analogues, and has broad market prospects and important social significance.

[0159] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. The use of insulin and its analogues in the preparation of drugs for the prevention or treatment of Alzheimer's disease.

2. Application of insulin and its analogues in the preparation of drugs to increase plasma insulin levels in Alzheimer's disease.

3. Application of insulin and its analogues in the preparation of drugs to improve cognitive impairment in Alzheimer's disease.

4. Application of insulin and its analogues in the preparation of drugs to improve brain microvascular lesions in Alzheimer's disease.

5. Application of insulin and its analogues in the preparation of drugs to restore brain glucose metabolism function in Alzheimer's disease.

6. Application of insulin and its analogues in the preparation of drugs to reduce the inflammatory response in Alzheimer's disease.

7. The application according to any one of claims 1-6, characterized in that, The insulin includes at least one of human insulin, glargine insulin, aspart insulin, lispro insulin, glutathione insulin, detemir insulin, and degludec insulin.

8. The application according to any one of claims 1-6, characterized in that, The administration methods of the drug include intravenous injection, intraperitoneal injection, and subcutaneous injection; And / or, the dosage of said insulin and its analogues is 0.5 U / kg - 2.0 U / kg.

9. A drug that can prevent or treat Alzheimer's disease, and / or increase plasma insulin levels in Alzheimer's disease, and / or improve cognitive impairment in Alzheimer's disease, and / or improve microvascular lesions in the brain in Alzheimer's disease, and / or reduce inflammatory responses in Alzheimer's disease, characterized in that, The drugs include insulin and its analogues.

10. The medicament according to claim 9, characterized in that, The insulin includes at least one of human insulin, glargine insulin, aspart insulin, lispro insulin, glutathione insulin, detemir insulin, and degludec insulin; And / or, the drug may also include one or more pharmaceutically acceptable excipients.

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