Pharmaceutical application of GLP-1 receptor stimulant semeglutide

Abstract

The invention relates to a pharmaceutical application of a GLP-1 receptor stimulant semeglutide, in particular to a novel pharmaceutical application of the GLP-1 receptor stimulant semeglutide, namely the semeglutide regulates hippocampal mitochondrial kinetics and mitochondrial autophagy balance by activating a phosphatidylinositol 3-kinase (PI3K) / protein kinase B (Akt) signal channel, so that the GLP-1 receptor stimulant semeglutide can be used for treating the hippocampal mitochondrial autophagy. The glial cell phenotype is regulated to be converted from a proinflammatory state to an anti-inflammatory state, and the cognitive impairment is improved. The invention determines a new medicine application method for linking the semeglutide with hippocampal pathology reduction and cognitive function recovery, and has important significance on treatment of VaD.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and particularly to a pharmaceutical use of the GLP-1 receptor agonist semaglutide. Background Art

[0002] Vascular dementia (VaD) is the most common cause of dementia after Alzheimer's disease (AD). It is associated with insufficient cerebral perfusion, leading to cerebrovascular damage and dysfunction. However, different from AD, there is currently no drug for the treatment of VaD. The current drug treatments for VaD mainly include non-competitive N-methyl-D-aspartic acid (NMDA) receptor antagonists (memantine) and cholinesterase inhibitors (donepezil). These two drugs are used to treat AD, but the efficacy is limited. The reason for testing these drugs in VaD is mainly based on the suggestive evidence of overlap between the two diseases in neurochemistry and neuropathology. Improving microcirculation, protecting nerves, and traditional drug treatments are also widely used in clinical practice. There is no conclusive evidence that these treatments delay the progression of dementia or improve symptoms. Compared with other dementias, there is less research on VaD and its mechanisms, and there is also a lack of treatment methods directly targeting its pathological mechanisms. Obviously, further pathophysiological research is needed to study the mechanisms leading to or accelerating cognitive deficits and to develop treatment methods targeting these mechanisms.

[0003] Glucagon-like peptide (GLP-1) and GLP-1 receptor agonists (GLP-1RAs) used to treat type 2 diabetes mellitus (T2DM) have neuroprotective effects in various neurodegenerative disease models, including ischemia, stroke, AD, Parkinson's disease, peripheral neuropathy, multiple sclerosis, and amyotrophic lateral sclerosis. Semaglutide is a long-acting GLP-1RA that allows once-weekly dosing. Semaglutide has been found to protect the integrity of the blood-brain barrier (BBB), reduce neuronal death, and thus play a role in protecting nerves and possibly reducing the risk of neurodegenerative diseases such as AD. Summary of the Invention

[0004] The present invention aims to solve the problem that there are no drugs and new uses targeting mitochondrial function for the treatment of vascular dementia in clinical treatment.

[0005] The object of the present invention is to newly explore the traditional pharmaceutical use of the GLP-1 receptor agonist semaglutide in neuroprotection and apply it to the field of drugs for vascular dementia, which is an innovation of a new drug use.

[0006] The present invention provides a pharmaceutical use of the GLP-1 receptor agonist semaglutide that can achieve the promotion of cognitive function recovery and target mitochondrial function and mitophagy.

[0007] There is no research on the role of semaglutide in vascular cognitive impairment (VCI) or VaD. The mechanism of action of semaglutide other than neuroprotection remains to be further explored and discovered. The experimental design and exploration process disclosed in the present invention: Application of semaglutide in improving cognitive dysfunction in a mouse model of vascular dementia by bilateral common carotid artery stenosis (BCAS).

[0008] Semaglutide affects the gene expression related to neuroinflammation, mitochondrial dynamics and mitophagy in the hippocampus of mice with vascular dementia.

[0009] Semaglutide inhibits the activation of microglia in the hippocampus of mice with vascular dementia and promotes the conversion of microglia to an anti-inflammatory phenotype.

[0010] Semaglutide inhibits the activation of astrocytes in the hippocampus of mice with vascular dementia and promotes the conversion of astrocytes to an anti-inflammatory phenotype.

[0011] Semaglutide inhibits excessive mitochondrial fission and promotes mitochondrial fusion in the hippocampus of mice with vascular dementia, maintaining the balance of mitochondrial dynamics.

[0012] Semaglutide inhibits excessive mitophagy in the hippocampus of mice with vascular dementia and restores the balance of mitophagy.

[0013] Application of semaglutide in activating the PI3K / AKT / mTOR signaling pathway in the hippocampus of mice with vascular dementia, inhibiting apoptosis, and reducing insulin resistance. Description of the Drawings

[0014] Figure 1 Shows the model of mice with vascular dementia: (a) Schematic diagram of the preparation of the model of mice with vascular dementia, (b) Grouping diagram of the application of semaglutide in the model of mice with vascular dementia.

[0015] Figure 2 Shows the effect of semaglutide on cerebral blood flow in mice with vascular dementia.

[0016] Figure 3 Shows the effect of semaglutide on mice with vascular dementia: (a, b) Show the effect of semaglutide on the motor ability of mice with vascular dementia; (c) Shows the recognition and memory of novel objects by mice with vascular dementia treated with semaglutide; (d) Shows the effect of semaglutide on working memory in mice with vascular dementia; (e-f) Show the effect of semaglutide on spatial learning and memory in mice with vascular dementia.

[0017] Figure 4 Shows the effect of semaglutide on gene expression such as mitochondrial dynamics and mitophagy.

[0018] Figure 5 Shows the effect of semaglutide on the gene GO pathway.

[0019] Figure 6 Shows the effect of semaglutide on glial cell expression: (a, c) show the effect of semaglutide on the expression of astrocytes in the hippocampus; (b, d) show the effect of semaglutide on the expression of microglia in the hippocampus.

[0020] Figure 7 Shows the effect of semaglutide on receptor expression: (a, d) show the effect of semaglutide on the expression of glucagon-like peptide receptor (GLP-1R) in astrocytes; (b, e) show the effect of semaglutide on the expression of GLP-1R in microglia; (c, f) show the effect of semaglutide on the expression of -GLP-1R in neurons; (g) shows the effect of semaglutide on the expression of GLP-1R in hippocampal tissue.

[0021] Figure 8 Shows the effect of semaglutide on the expression of markers in microglia: (a, c) show the effect of semaglutide on the expression of the pro-inflammatory phenotype marker CD68 in microglia; (b, d) show the effect of semaglutide on the expression of the pro-inflammatory phenotype marker CD206 in microglia; (e) shows the effect of semaglutide on the expression of the CD68 gene.

[0022] Figure 9 Shows the effect of semaglutide on the pro-inflammatory phenotype markers in astrocytes: (a, c) show the effect of semaglutide on the expression of the pro-inflammatory phenotype marker C3 in astrocytes; (b, d) show the effect of semaglutide on the expression of the pro-inflammatory phenotype marker S100a10 in astrocytes.

[0023] Figure 10 Shows the effect of semaglutide on the expression of genes related to mitochondrial dynamics.

[0024] Figure 11 Shows the effect of semaglutide on the expression of insulin receptor (p-IRS / IRS1) and PI3K / AKT / mTOR signaling pathway.

[0025] Figure 12 Shows the effect of semaglutide on the expression of genes related to mitophagy.

[0026] Figure 13 Shows the effect of semaglutide on the expression of apoptosis-related genes and proteins.

[0027] Figure 14 Shows the results of western blot detection images of all detected protein bands in this experiment.

[0028] Figure 15 It is the qRT-PCR primer sequence. Detailed implementation manners

[0029] The present invention provides a new pharmaceutical use of the GLP-1 receptor agonist semaglutide, that is, semaglutide regulates mitochondrial dynamics and mitochondrial autophagy balance in the hippocampal region by activating the phosphatidylinositol 3-kinase (PI3K) / protein kinase B (Akt) signaling pathway, regulates the transformation of glial cell phenotypes from a pro-inflammatory state to an anti-inflammatory state, and improves cognitive dysfunction. This study has determined a new application method of the drug that links semaglutide with the reduction of hippocampal pathology and the restoration of cognitive function, which is of great significance for the treatment of VaD.

[0030] <Experiment> 1 Preparation of experimental model A vascular dementia mouse model was prepared using the bilateral common carotid artery stenosis method (BCAS), see Figure 1 (a). Semaglutide (dissolved in physiological saline) was injected intraperitoneally. The mice were randomly divided into a control group and a BCAS group: the sham operation group was injected with an equal amount of physiological saline (Sham + vehicle, Veh), the sham operation + semaglutide group was injected with semaglutide once every 2 days (Sham + Sema), the BCAS group was injected with an equal amount of physiological saline (BCAS + Veh), and the BCAS + Sema group (BCAS + Sema). Cerebral blood flow (CBF) was measured before surgery, 2 h after surgery, 28 days after surgery, and 60 days after surgery. Behavioral tests were performed 28 days after surgery, including the open field test (OFT), novel object recognition (NOR), Y maze, and Morris water maze (MWM). Mice with cognitive decline were randomly divided into a physiological saline control group and a semaglutide group. Sham-operated mice were randomly divided into a physiological saline control group and a semaglutide administration group. Mice in the semaglutide group were injected intraperitoneally with semaglutide (30 nmol / kg), and mice in the control group were injected with an equal amount of physiological saline. From the 28th day to the 60th day after surgery, injections were continuously given once every 2 days. All animals were raised to the 60th day after BCAS or sham operation for subsequent analyses, including behavioral tests, ribonucleic acid (RNA) sequencing (RNA-seq), immunofluorescence, polymerase chain reaction (PCR), and western blot analysis, see Figure 1 (b).

[0031] The following further designed multiple <Experiments> to verify and support the exploration and discovery results of the new application of the drug of the present invention from different levels.

[0032] <Experiment> 2 Mice in the saline (Veh) or semaglutide (Sema) treatment groups (n = 3 per group) had their CBF measured before BCAS surgery (baseline), at 2 h, 28 days, and 60 days. The skull was exposed through a midline skin incision, and a high-resolution laser speckle flowmeter was used to select regions of interest (ROIs) between lambda and bregma in both hemispheres for whole-brain perfusion. The CBF changes in the acquired images were analyzed using a specific PIMSoft program (Peried Inc., Sweden). BCAS caused a decrease in CBF at 2 h postoperatively compared to the baseline level in both the BCAS + Veh group and the BCAS + Sema group. Over time, the CBF in the mice was detected to gradually recover at 28 days and 60 days. However, there was no statistically significant difference in the effect of semaglutide treatment on CBF at 2 h, 28 days, and 60 days post-BCAS compared to the BCAS + Veh control group.

[0033] It can be seen from Figure 2 that semaglutide has no significant effect on cerebral blood flow in BCAS mice.

[0034] <Experiment> 3 Behavioral tests The open-field experiment was used to evaluate the locomotor behavior of mice. The open-field device consisted of a square area (40 × 40 cm), and the 40-cm-high walls were made of white acrylic plastic sheets. The locomotion of the mice was tracked by a custom-made image-tracking program connected to an overhead camera. The total locomotion distance (cm) and average speed (mm / s) were automatically collected by the tracking software. Data were recorded for 20 min on the 28th and 60th days postoperatively (sham surgery or BCAS). On the 60th day, the open-field experiment measured the locomotor ability of the mice, and there was no statistically significant difference in the locomotion distance ( Figure 3 (a)) and speed ( Figure 3 (b)) between the groups. This indicates that semaglutide treatment has no significant effect on the locomotor ability of BCAS or sham-surgery mice.

[0035] The novel object recognition experiment was used to evaluate various aspects of mouse memory and learning. In this study, mice were required to acclimate to a testing arena of 40 × 40 × 40 cm for 20 minutes. The next day, the mice underwent sampling and testing. During sampling, each mouse had 20 minutes to study two identical objects, which were placed diagonally at equal distances from the center. Two hours later, during the testing process, one of the sample objects was replaced by a novel object, and the mice explored for another 20 minutes. Software was used to record the behavioral trajectories and the time spent exploring these objects. The novel object preference ratio was calculated by dividing the total time spent exploring the two objects during the testing process by the total time spent exploring the novel object. Data were recorded on the 28th and 60th days after surgery (sham or BCAS). On the 60th day, compared with BCAS + Veh mice, mice injected with semaglutide spent more time studying the novel object ( Figure 3 (c)), indicating an improvement in cognitive function in the NOR test.

[0036] Spontaneous alternation activity in the Y-maze test was recorded to evaluate spatial working memory. Each mouse had 8 minutes to freely explore the Y-maze arena (arms 30 cm long, 10 cm wide, and 20 cm high) and started from the same arm. Entries into the arms were recorded during the test (the criterion was that all four paws were within the perimeter of the arm). The spontaneous alternation rate (%) = [number of triads (entries into three different arms in succession) / (total number of entries - 2) * 100]. Data were recorded on the 28th and 60th days after surgery (sham or BCAS). On the 60th day, compared with the control group, semaglutide significantly increased the spontaneous alternation rate in BCAS mice ( Figure 3 (d)), indicating an improvement in the working memory impairment in BCAS mice.

[0037] The MWM was used to evaluate spatial learning and memory. The maze consisted of a gray plastic circular pool (50 cm high and 120 cm in diameter) filled with water to a depth of 45 cm. On day 1, the mice underwent three 60-second trials, each separated by approximately 45 minutes, to find the platform (1 cm above the water surface and 10 cm in diameter). From day 2 to day 5, spatial learning ability was examined through navigation trials. All mice were tested 4 times a day, each separated by 15 minutes, for 4 consecutive days. During the trials, the average swimming speed and escape latency were recorded. On day 6, spatial memory was evaluated through a probe test. Software was used to video-track the activity trajectories of the mice. The proportion of the swimming time in the target quadrant on the probe day was recorded and analyzed. Data were recorded on the 28th and 60th days after surgery (sham or BCAS). In the navigation trials starting on the 60th day, the escape latency of BCAS mice decreased ( Figure 3 (e)), and the residence time in the platform quadrant increased on the probe day after semaglutide treatment ( Figure 3(f)), indicating the improvement of spatial learning and memory.

[0038] <Experiment> 4 Investigation on signal pathways and regulation Extract total RNA from the hippocampus of mice. Detect the purity and quantity of RNA, and then prepare an RNA-seq library for mRNA sequencing. Collect and analyze the differentially expressed genes (DEGs) between BCAS + Sema and BCAS + Veh. The criteria for identifying DEGs were fold change ≥ 2 and p-value < 0.05. Principal component analysis (PCA) was used to observe the main factors leading to sample variation. The volcano plot showed the overall distribution of DEGs. Gene ontology (GO) analysis was used to enrich the functions of DEGs. Compared with the BCAS + Veh group, there were relatively fewer DEGs in the BCAS + Sema group (113 DEGs were downregulated and 42 DEGs were upregulated). After treatment with semaglutide, pro-inflammatory genes (CD44, CD86, C3, Cxcl1, and Il12a) and apoptotic genes (Bax and Bak) decreased, while anti-inflammatory genes (Il6, S100a10, Cx3cr1, and CD36) and mitophagy genes (Map1lc3b, Sqstm1, and Atg9a) increased. In addition, the genes related to mitochondrial dynamic fusion and fission (Mfn1, Mfn2, OPA1, and Dnm1l) - expression was normalized ( Figure 4 ). To gain an in-depth understanding of the impact of DEGs on biological functions, GO enrichment analysis was performed, and the top 30 GO terms (p < 0.05, log2 FC > 1) were given. The GO enrichment of DEGs was divided into three types, namely cellular components (CCs), molecular functions (MFs), and biological processes (BPs). It was found that compared with BCAS + Veh mice, BCAS + Sema mice had significantly enhanced positive regulation of the extracellular signal-regulated kinase 1 (ERK1) and ERK2 cascades, immune response, mitochondrial fusion, mitochondrial morphogenesis, and neuropeptide signaling pathways in BPs, axons, neuronal cell bodies, and neuronal projections in CCs, and signal receptor binding, growth factor activity, oxygen binding, transmembrane signal receptor, and G protein-coupled receptor (GPCF) activity in MFs ( Figure 5 ).

[0039] <Experiment> 5. The brains of the mouse models were cut into 10-μm-thick slices using a cryostat for immunofluorescence staining. After permeabilization with Triton X-100 for 20 minutes, the brain slices were blocked with 3% donkey serum for half an hour and incubated overnight at 4 °C with primary antibodies (GFAP, Iba-1). Subsequently, the slices were rinsed with PBS (3 × 10 min) and incubated with secondary antibodies for 1 hour at room temperature in the dark. After staining with 4′,6-diamidino-2-phenylindole (DAPI), the samples were scanned using a confocal laser scanning microscope. Cell counting was performed by two independent researchers. Three to five frozen sections were prepared from the hippocampal tissues of each mouse model brain, and three to five images were taken at a 20× objective magnification for each section. The positively stained cells in the hippocampal cornu ammonis (CA) 1, CA2, CA3, and dentate gyrus (DG) regions were quantified. The data were expressed as the mean number of positively stained cells per field of view. In the semaglutide-treated group of BCAS mice, the number of GFAP-immunopositive astrocytes ( Figure 6 (a, c)) and Iba-1-immunostained activated microglia ( Figure 6 (b, d)) in the hippocampus decreased.

[0040] <Experiment> 6 Based on <Experiment> 5, further experiments were conducted to explore To determine the potential mechanism of action of semaglutide, western blot was used to detect GLP-1R levels. After the mouse models inhaled a lethal dose of carbon dioxide and died, the brain tissues were collected (n = 5-8 per group). The hippocampal tissues were homogenized by sonication with 300 μL of radioimmunoprecipitation assay (RIPA) buffer, protease inhibitor mixture, and phosphatase inhibitor. The protein concentration was determined using the bicinchoninic acid (BCA) protein assay. Equal concentrations and volumes of proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. Subsequently, the separated proteins were transferred to polyvinylidene fluoride (PVDF) membranes using the wet transfer method. After blocking with 5% non-fat dry milk or bovine serum albumin (BSA) for 1 hour, the membranes were incubated with the primary antibody (GLP-1R) in 3% milk or BSA in tris-buffered saline with Tween 20 (TBST; 50 mM tris and 150 mM sodium chloride (NaCl), 0.1% Tween 20, pH 7.6) at 4 °C overnight. Subsequently, the membranes were incubated with the specific secondary antibody for 1 hour. The Bio-Rad Gel Doc XR+ imaging system was used to detect specific protein bands. The detection image results of all protein bands are shown in Figure 14 ( Figure 14 a The expression of GLP-1R and proteins related to mitochondrial dynamics (MFN1, MFN2, DRP1, p-DRP1, FIS1, and OPA1), Figure 14b Expression of PI3K / Akt / mTOR signaling pathway and autophagy-related proteins (P62 and LC3B), Figure 14 c Expression of autophagy-related proteins (Atg7 and Atg5) and apoptosis-related proteins (BCL-2, BAX, BCL-X and BAK).

[0041] Compared with the control group, western blot detection showed that the expression level of GLP-1R protein in the semaglutide administration group increased, suggesting that semaglutide acts through GLP-1R ( Figure 7 g, Figure 14 a). Meanwhile, immunofluorescence staining images showed the co-expression of GLP-1R with GFAP, Iba1, and NeuN in the hippocampal tissue (the same method as in <Experiment> 5). GLP-1R was highly expressed in microglia ( Figure 7 (b, e)), and lowly expressed in astrocytes ( Figure 7 (a, d)) and neurons ( Figure 7 (c, f)), indicating that the action site of semaglutide is mainly in hippocampal glial cells.

[0042] <Experiment> 7 Based on <Experiment> 6, further experiments were conducted for verification The effect of semaglutide on microglial phenotype was detected by immunofluorescence staining (the same method as in <Experiment> 5). Immunofluorescence staining images showed the co-localization of Iba-1 with its polarization markers cluster of differentiation 68 (CD86) and CD206, indicating the activation of M1 (CD86-positive) and M2 (CD206-positive) microglia in the hippocampal region after BCAS compared with the sham-operated control group. Compared with BCAS + Veh mice, the number of M1 (CD86-positive) microglia in the hippocampus of BCAS + Sema mice decreased ( Figure 8 (a, c)), and the number of M2 (CD206-positive) microglia increased ( Figure 8 (b, d)). In addition, the expression of the CD86 gene was detected by PCR. TRIzol reagent was used to extract total RNA. Complementary deoxyribonucleic acid (cDNA) synthesis was performed on PrimeScript™ RT Master Mix according to the manufacturer's instructions. Reverse transcription PCR (RT-PCR) was performed using the StepOnePlus PCR system in a 20 µL reaction mixture with TB Green® Premix Ex Taq™ II. The primer sequences are shown in Table 1 ( Figure 15 ). The results showed that the expression of the CD86 gene increased after BCAS surgery, while the expression of the CD86 gene decreased after administration of semaglutide ( Figure 8 (e)). These results indicate that microglia switch to an anti-inflammatory phenotype after semaglutide treatment.

[0043] Experiment 8 Based on Experiment 7, further experimental verification was carried out The effect of semaglutide on the phenotype of astrocytes was detected by immunofluorescence staining (the method was the same as in Experiment 5). Immunofluorescence images showed the co-localization of GFAP with its polarization markers C3 and S100A10, indicating the activation of A1 and A2 astrocytes (C3-positive and S100A10-positive, respectively) in the hippocampus after BCAS. In addition, compared with BCAS + Veh mice, the number of A1 (C3-positive) astrocytes in the hippocampus of BCAS+ Sema mice decreased ( Figure 9 (a, c)), while the number of A2 (S100A10-positive) astrocytes increased ( Figure 9 (b, d)). These results indicate that astrocytes shift to an anti-inflammatory phenotype after semaglutide treatment.

[0044] Experiment 9 Following Experiment 4, verification of mitochondrial dynamics was carried out First, the expression of mitochondrial dynamics-related genes Mfn1, Mfn2, Dnm1, FIS1, and OPA1 was detected by PCR (the method was the same as in Experiment 7). Compared with the sham-operated control group, the expression levels of mitochondrial fission-related genes Dnm1 and FIS1 increased in the hippocampal tissues of BCAS-operated mice ( Figure 10 (a-b)), while the expression levels of mitochondrial fusion-related genes Mfn1, Mfn2, and OPA1 decreased ( Figure 10 (c-f)). However, after semaglutide treatment, this trend was reversed ( Figure 10 (a-f)). Then, the expression of DRP1, OPA1, p-DRP1 (616), Mfn1, Mfn2, and FIS1 proteins was detected by western blot (the method was the same as in Experiment 6). The levels of p-DRP1(616) / DRP1 and FIS1 increased in the hippocampus of BCAS mice ( Figure 10 (g-h), Figure 14 a), but the levels of Mfn1, Mfn2, and OPA1 were lower than those in sham-operated mice ( Figure 10 (i-e), Figure 14 a). By differentially regulating fission (FIS1 and DRP1) and fusion (Mfn1, Mfn2, and OPA1) in the hippocampus of BCAS mice, the mitochondrial dynamic balance tilted towards fission, resulting in mitochondrial fragmentation. However, the results showed that semaglutide treatment reduced the fission DRP1 and FIS1 proteins ( Figure 10 (g-h), Figure 14 a) and increased the fusion Mfn2, Mfn1, and OPA1 proteins ( Figure 10(i.e.), Figure 14 The expression of a) was used to prevent mitochondrial fragmentation.

[0045] <Experiment> 10 Following <Experiment> 4, verification of the signaling pathway was carried out. The PI3K / Akt / mTOR signaling pathway was detected by western blot (the method was the same as in <Experiment> 6). The results showed that compared with the sham operation group, the expression level of IRS1 in the BCAS group increased, but the difference between groups was not statistically significant. In addition, the p-IRS1 (Ser612) / IRS1 in the BCAS + Veh group was significantly lower than that in the BCAS + Veh group ( Figure 11 a, Figure 14 b). After administration of semaglutide, the expressions of p-PI3K / PI3K, p-Akt / Akt, and p-mTOR / mTOR in BCAS mice were all significantly increased ( Figure 11 b-d, Figure 14 b).

[0046] <Experiment> 11 Following <Experiment> 4, verification of the signaling pathway was carried out. The expression levels of Sqstm1 and Map1lc3b genes were detected by PCR (the method was the same as in <Experiment> 7). The results showed that the expression of the Sqstm1 gene increased in the hippocampal tissue of BCAS mice ( Figure 12 a), and the expression of the Map1lc3b gene decreased ( Figure 12 b). In fact, compared with the control, semaglutide treatment decreased the Sqstm1 level ( Figure 12 a), and increased the Map1lc3b level ( Figure 12 b). In addition, the levels of autophagy-related proteins LC3-B, SQSTM / p62, ATG5, and ATG7 were detected by western blot (the method was the same as in <Experiment> 6). The levels of mitochondrial autophagy-related proteins LC3-B, ATG5, and ATG7 in the hippocampus of VaD mice decreased ( Figure 12 d-f, Figure 14 b, c), and the level of SQSTM / p62 increased (c), indicating a decrease in autophagy after BCAS. In fact, semaglutide reversed this trend, indicating that semaglutide promoted the recovery of mitochondrial autophagy to normal levels ( Figure 12 , Figure 14 b, c).

[0047] <Experiment> 12 Following <Experiment> 4, verification of the signaling pathway was carried out. The expression levels of Bcl2 and Bax genes were detected by PCR (the same method as in <Experiment> 7). The results showed that the expression level of the anti-apoptotic gene Bcl2 decreased and the expression level of the pro-apoptotic gene Bax increased after BCAS ( Figure 13 a-b, Figure 14 c), and this change was reversed after treatment with semaglutide. In addition, the expressions of Bcl-2, Bax, Bcl-XL, and Bak proteins were detected by western blot (the same method as in <Experiment> 6). The anti-apoptotic proteins Bcl-2 and BCL-X decreased after BCAS ( Figure 13 c-d), and the pro-apoptotic proteins Bax and Bak increased ( Figure 13 e-f), which was consistent with the gene expression. After treatment with semaglutide, this trend was reversed, suggesting that semaglutide plays a protective role by inhibiting apoptosis ( Figure 13 , Figure 14 c).

Claims

1. Pharmaceutical use of semaglutide, a GLP-1 receptor agonist.

2. The pharmaceutical use according to claim 1, wherein It is a drug in the field of vascular dementia.

3. The pharmaceutical use according to claim 2, wherein Specifically, it is a new use of a drug for targeting mitochondrial function to treat vascular dementia.

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