Application of neurogenic exosome synaptophysin in the preparation of products for the treatment of non-dementia obstructive sleep apnea

Abstract

The present invention relates to the field of biomedicine, and in particular to the use of neurogenic exosomal synaptophysin in the preparation of a product for treating non-dementia obstructive sleep apnea. By detecting the expression level of the biomarker neurogenic exosomal synaptophysin in a sample, it is possible to diagnose whether a subject has non-dementia obstructive sleep apnea, enabling early intervention and effective treatment.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of neurogenic exosome synaptophysin in the preparation of products for treating non-dementia obstructive sleep apnea. Background Art

[0002] Obstructive sleep apnea (OSA) is the most common sleep disorder, with a prevalence of approximately 9-38%. It is characterized by recurrent breathing pauses, intermittent hypoxia, sleep fragmentation, and daytime sleepiness. OSA is a significant risk factor for cognitive impairment and even dementia, with patients with OSA having a 30% increased risk of cognitive impairment. Cognitive impairment in OSA patients is typically associated with impairments in attention, memory, executive function, psychomotor speed, and visuospatial and structural abilities, with less involvement of language skills.

[0003] Intermittent hypoxia can lead to adverse reactions such as oxidative stress, inflammation, synaptic dysfunction, and overactivation of the sympathetic nervous system. Increased reactive oxygen species and decreased antioxidant capacity directly affect synaptic activity and are a key mechanism of cognitive impairment. Currently, the role of altered expression of synaptic proteins in neuronal-derived exosomes (NDEs) in cognitive impairment in patients with non-dementia OSA has not been studied. Therefore, it is important to clarify the diagnostic value of synaptic proteins in NDEs in cognitive impairment in patients with non-dementia OSA, thereby providing a foundation for the development of products and drugs to treat non-dementia OSA. Summary of the Invention

[0004] The present invention provides the use of neurogenic exosome synaptophysin in the preparation of a product for treating non-dementia obstructive sleep apnea. By detecting the expression level of the biomarker neurogenic exosome synaptophysin in a sample, it is possible to diagnose whether a subject has non-dementia obstructive sleep apnea, intervene early and carry out effective treatment, thereby solving the problems existing in the prior art.

[0005] The present invention provides the following technical solutions:

[0006] The invention relates to the use of a reagent for detecting a biomarker in a sample in the preparation of a product for diagnosing non-dementia obstructive sleep apnea disease, wherein the biomarker is a neurogenic exosomal synaptophysin and the sample is peripheral venous blood plasma.

[0007] Furthermore, the neurogenic exosomal synaptic proteins include synaptic proteins GAP43, SNAP25, and / or NRGN.

[0008] Furthermore, the reagent is a reagent for detecting changes in the expression level of neurogenic exosome synaptic proteins.

[0009] Further, when the expression of synaptic proteins GAP43, SNAP25, and / or NRGN is elevated, the subject suffers from non-dementia obstructive sleep apnea disease.

[0010] Furthermore, when the expression of synaptic proteins GAP43, SNAP25, and NRGN was elevated, the subjects had non-dementia obstructive sleep apnea.

[0011] Furthermore, the reagent includes an antibody or an antibody fragment.

[0012] Furthermore, the reagent is an antibody or antibody fragment that can specifically bind to the neurogenic exosomal synaptic proteins GAP43, SNAP25, and / or NRGN.

[0013] Furthermore, the product includes a chip or a kit.

[0014] Furthermore, the kit is an immunohistochemistry kit or a protein blotting kit.

[0015] The present invention also provides the following technical solutions:

[0016] The use of a substance for detecting the expression levels of neurogenic exosomal synaptic proteins GAP43, SNAP25, and / or NRGN in a sample in screening drugs for treating non-dementia obstructive sleep apnea.

[0017] Beneficial effects of the present invention:

[0018] The present invention obtained that the neurogenic exosomal synaptic proteins GAP43, SNAP25, and NRGN are elevated in non-dementia OSA patients with cognitive impairment, and are significantly higher than those in the healthy control group. The correlation between the expression of the above synaptic proteins and cognitive impairment in non-dementia OSA patients was determined. This is contrary to the expression changes of individual proteins related to exosome synaptic growth in AD reported in the prior art. This study facilitates early auxiliary diagnosis and parallel intervention of OSA, reducing the further development of this type of disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the exosome concentration of synaptic protein GAP43 among the three groups in the present experiment;

[0020] Figure 2 is the exosome concentration of synaptic protein SNAP25 among the three groups in the present experiment;

[0021] Figure 3 The exosome concentrations of synaptic protein NRGN among the three groups in the present experiment;

[0022] Figure 4The changes in the exosome concentration of synaptic protein GAP43 in OSA patients before and after the intervention treatment of the present invention;

[0023] Figure 5 The changes in the exosome concentration of synaptic protein SNAP25 in OSA patients before and after the intervention treatment of the present invention;

[0024] Figure 6 The changes in the exosome concentration of synaptic protein NRGN in OSA patients before and after the intervention treatment of the present invention;

[0025] Figure 7 MoCA scores of OSA patients before and after the intervention treatment of the present invention;

[0026] Figure 8 This is the ROC curve of the diagnostic value of the synaptic protein NDE level for cognitive impairment in non-dementia OSA patients of the present invention. DETAILED DESCRIPTION

[0027] In order to clearly illustrate the technical features of this solution, the present invention is described in detail below through specific implementation methods in conjunction with the accompanying drawings.

[0028] Unless otherwise specified, the instruments, reagents, and materials used in the following examples are all conventional instruments, reagents, and materials available in the prior art and can be obtained through regular commercial channels. The experimental methods and detection methods used in the following examples are all conventional experimental methods and detection methods available in the prior art, unless otherwise specified.

[0029] 1. Detection of the expression level of neurogenic exosome synaptic proteins in subject samples

[0030] 1.1 Materials and Methods

[0031] 1.1.1 Inclusion criteria: Patients met the diagnostic criteria for OSA in the 2018 Multidisciplinary Guidelines for the Diagnosis and Treatment of Obstructive Sleep Apnea in Adults: that is, meeting the following (A+B) or C.

[0032] A: At least one of the following occurs: ① The patient complains of drowsiness, non-restorative sleep, fatigue, or insomnia; ② Awakening from sleep due to breath holding or gasping; ③ Roommates or other witnesses report that the patient has habitual snoring, respiratory arrest, or both during sleep; ④ Diagnosed hypertension, mood disorders, cognitive impairment, coronary heart disease, cerebrovascular disease, congestive heart failure, atrial fibrillation, or type 2 diabetes.

[0033] B: Polysomnography confirms respiratory events ≥ 5 times / hour during the monitoring period, including obstructive apnea, mixed apnea, hypopnea, and respiratory effort-related arousals.

[0034] C: Polysomnography confirmed respiratory events ≥15 times / hour during the monitoring period, including obstructive apnea, mixed apnea, hypopnea, and respiratory effort-related arousals; (2) aged 35 to 80 years; (3) no previous diagnosis of OSA and no related treatment; (4) no neuropsychological evaluation in the past year and no history of major surgery or trauma in the past 3 months.

[0035] 1.1.2 Exclusion criteria: (1) Patients with any history of neurological or psychiatric diseases that affect cognitive function, including stroke, intracranial infection, brain trauma, hydrocephalus, epilepsy, brain tumors, neurodegenerative diseases (such as AD, Parkinson's disease, multiple system atrophy and Lewy body dementia), other sleep disorders (such as narcolepsy and restless legs syndrome), schizophrenia and major depressive disorder; (2) Patients using medications known to affect cognition or sleep, such as benzodiazepines, antihistamines, tricyclic antidepressants, donepezil and citicoline; (3) Patients with cognitive impairment without OSA; (4) Abnormal thyroid function, including hypothyroidism and hyperthyroidism; (5) Severe cardiopulmonary insufficiency, liver and kidney failure; (6) Autoimmune system diseases, including systemic lupus erythematosus and Sjögren's syndrome; (7) Pregnant women.

[0036] 1.1.3 Interventional treatment: Treatment recommendations were given to moderate to severe OSA patients. Patients with moderate to severe OSA who agreed to use continuous positive airway pressure (CPAP) were included in the longitudinal study. Inclusion criteria included: (1) good compliance with CPAP treatment, using the ventilator for more than 4 hours per night and more than 5 nights per week; (2) CPAP treatment was effective, as reported by the ventilator software (AHI < 5 times / hour); and (3) adherence to CPAP treatment for at least 1 year.

[0037] 1.1.4 Cognitive assessment: A MoCA cutoff score of 26 was used, and the revised Mayo Clinic criteria were used to diagnose mild cognitive impairment (MCI), which includes (1) subjective (including patient or informed) cognitive impairment; (2) objective impairment in one or more cognitive domains; (3) mild impairment in complex instrumental daily abilities, but the ability to maintain independent daily living abilities; and (4) not yet reaching the diagnosis of dementia. All subjects were divided into three groups based on the apnea-hypopnea index (AHI) and MoCA score: OSA+MCI group (AHI ≥ 5, MoCA score < 26); OSA-MCI group (AHI ≥ 5, MoCA ≥ 26); and control group (AHI < 5, MoCA ≥ 26).

[0038] The subjects were patients enrolled from the Sleep Medicine Center of Weihai Municipal Hospital from June 2020 to December 2021, and the subjects in the control group were recruited from healthy people in the community.

[0039] 1.2 Detection indicators: Levels of synaptic proteins GAP43, SNAP25, and NRGN in neuron-derived exosomes (NDEs).

[0040] The expression levels of the above-mentioned synaptic proteins GAP43, SNAP25, and NRGN were detected as follows:

[0041] Venous blood was drawn from all subjects between 6:00 and 7:00 AM on the morning of the second day after sleep monitoring and stored in polypropylene tubes containing EDTA. Within 30 minutes after blood draw, the blood samples were centrifuged at 4000 × g for 10 minutes to obtain plasma and isolate specific NDEs. The experimental procedures are described as follows:

[0042] 0.5 ml of plasma was taken and incubated with thromboplastin-D at room temperature for 60 minutes. Then, 0.35 ml of calcium- and magnesium-free Dulbecco's phosphate buffer and protease inhibitor cocktail were added. The mixture was centrifuged at 3000 × g for 20 minutes at 4°C. The supernatant was incubated with ExoQuick exosome precipitation solution at 4°C for 1 hour.

[0043] The cells were centrifuged at 1500 × g for 30 minutes at 4°C, and the pellet was resuspended in 250 μl of DPBS. Each exosome suspension was added with 100 μl of 3% bovine serum albumin (BSA) and incubated with 3 μl of rabbit anti-L1 cell adhesion molecule (L1CAM) antibody at 4°C for 2 hours.

[0044] Add 25 μl of streptavidin-resin and 50 μl of 3% BSA. Centrifuge at 400 × g for 10 minutes at 4°C. Remove the supernatant and resuspend the pellet in 50 μl of 0.05 M glycine-HCl (pH 3.0). Vortex and mix for 10 minutes. Add 0.4 ml of Mammalian Protein Extraction Reagent (M-PER) adjusted to pH 8.0 with 1 M Tris-HCl (pH 8.6). Incubate at 37°C for 10 minutes, then vortex and mix for 15 seconds. Store at -80°C.

[0045] Plasma NDEs levels of GAP43, SNAP25, and NRGN were measured using enzyme-linked immunosorbent assay (ELISA) kits. CD81 protein content was measured using an ELISA kit to normalize exosome content. The mean of all measured CD81 levels in each group was set to 1.00, and the relative value of each sample was used for normalization.

[0046] 1.3 Statistical Methods: Data were analyzed using SPSS 22.0, R 4.1.1, and GraphPad Prism 8.3.0. Two-tailed statistical tests were used, and P < 0.05 was considered statistically significant.

[0047] 1.4 Results

[0048] 1.4.1 Synaptic protein NDE levels among the three groups

[0049] In the OSA+MCI group (AHI≥5, MoCA<26), the NDE level of GAP43 was 3353.75±1312.31 pg / ml, the NDE level of SNAP25 was 1036.58±336.48 pg / ml, and the NDE level of NRGN was 2554.23±883.13 pg / ml. In the OSA-MCI group (AHI≥5, MoCA≥26), the NDE level of GAP43 was 2976.13±1022.59 pg / ml. , the NDE level of SNAP25 was 792.80±171.70pg / ml, and the NDE level of NRGN was 2273.95±630.53pg / ml; in the control group (AHI<5, MoCA≥26), the NDE level of GAP43 was 2699.01±450.54pg / ml, the NDE level of SNAP25 was 636.91±61.88pg / ml, and the NDE level of NRGN was 2091.85±363.63pg / ml.

[0050] See also Figure 1-Figure 3 Compared with the control group, the NDE levels of synaptic proteins GAP43, SNAP25, and NRGN in the OSA+MCI group and the OSA-MCI group were significantly increased, and there were also significant statistical differences in the NDE levels of synaptic proteins between the OSA+MCI group and the OSA-MCI group.

[0051] 1.4.2 Changes in MoCA scores and synaptic protein NDE levels in OSA patients before and after intervention

[0052] See also Figure 4-7, after 1 year of CPAP treatment, it was observed that the NDE levels of synaptic proteins GAP43, SNAP25, and NRGN in non-demented OSA patients were significantly decreased compared with baseline, and the MoCA levels were significantly improved.

[0053] 1.4.3 Diagnostic value of synaptic protein NDE levels for cognitive impairment in non-demented OSA patients

[0054] See also Figure 8 The NDE levels of synaptic proteins GAP43, SNAP25, and NRGN jointly predicted mild cognitive impairment in non-demented OSA patients. Receiver operating characteristic (ROC) curve analysis showed an area under the curve of 0.857. These results suggest that the NDE levels of synaptic proteins GAP43, SNAP25, and NRGN have a good diagnostic value for cognitive impairment in non-demented OSA patients.

[0055] The above specific implementation manner cannot be used as a limitation on the protection scope of the present invention. For those skilled in the art, any replacement, improvement or transformation made to the implementation manner of the present invention falls within the protection scope of the present invention.

[0056] Any matters not described in detail in the present invention are well-known technologies to those skilled in the art.

Claims

1. Use of a reagent for detecting biomarkers in a sample in the preparation of a product for diagnosing mild cognitive impairment in patients with non-dementia obstructive sleep apnea, characterized in that: The biomarkers are neurogenic exosomal synaptic proteins GAP43, SNAP25 and NRGN; and the sample is peripheral venous blood.

2. The use according to claim 1, characterized in that The reagent is a reagent for detecting changes in the expression levels of neurogenic exosome synaptic proteins GAP43, SNAP25 and NRGN.

3. The use according to claim 2, characterized in that When expression of the synaptic proteins GAP43, SNAP25, and NRGN was elevated, the subjects had non-dementia obstructive sleep apnea.

4. The use according to claim 1 or 2, characterized in that The reagent comprises an antibody or an antibody fragment.

5. The use according to claim 1, characterized in that The product includes a chip or a kit.

6. Application of substances that detect the expression levels of neurogenic exosomal synaptic proteins GAP43, SNAP25 and NRGN in peripheral venous blood samples in screening drugs for the treatment of mild cognitive impairment in patients with non-dementia obstructive sleep apnea.

Images