Application of inhibitors targeting the COLLAGEN pathway in the preparation of drugs for treating Alzheimer's disease associated with exposure to micro / nanoplastics particles.

Abstract

This invention discloses the application of an inhibitor targeting the COLLAGEN pathway in the treatment of Alzheimer's disease (AD) associated with exposure to micro / nanoparticle plastics (MNPs). The study shows that MNP exposure activates the COLLAGEN signaling pathway between astrocytes, microglia, and neurons, promoting abnormal cell communication mediated by COL1A1 / COL1A2-ITGA1 / ITGB1, leading to M1 polarization in microglia, activation of the neuronal p38-MAPK pathway, and Aβ deposition, thereby accelerating AD progression. By knocking down the ligand COL1A1 in astrocytes to block this pathway, neuroinflammation is significantly reduced, Aβ accumulation is inhibited, and cognitive function is improved. This invention provides a novel molecular target for the treatment of MNP-related AD.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and more specifically, this invention relates to the application of inhibitors targeting the COLLAGEN pathway in the preparation of drugs for treating Alzheimer's disease associated with exposure to micro / nanoplastics particles. Background Technology

[0002] Micro-nanoplastics (MNPs) are a class of emerging pollutants widely present in the environment, primarily originating from industrial emissions and degradation of plastic products. With increasing plastic pollution, MNPs can enter the human body through various pathways, including the food chain, drinking water, and air, accumulating in multiple organs, with the central nervous system being a significant target organ. A recent report in *The Lancet* indicates that particulate matter exposure is one of the modifiable key risk factors for Alzheimer's disease (AD). In recent years, particulate pollutants, especially the emerging pollutant MNPs, have gradually been recognized as important exogenous risk factors influencing the occurrence and progression of AD. Recent studies have reported significantly higher MNP concentrations in the brains of AD patients compared to healthy individuals. Animal experiments further demonstrate that long-term MNP exposure can lead to disruption of the blood-brain barrier integrity in mice, exacerbated neuroinflammatory responses in the hippocampus, and significant cognitive impairment and memory deficits. These findings provide direct evidence for MNPs as an important exogenous risk factor for AD. Alzheimer's disease (AD) is a neurodegenerative disease characterized by β-amyloid (Aβ) deposition, neuroinflammation, and neuronal loss, for which there is currently no effective treatment. Existing research suggests that mesenchymal nuclei (MNPs) may promote AD progression by inducing oxidative stress, neuroinflammation, and exacerbating Aβ accumulation. However, the specific molecular mechanisms by which MNPs promote AD development remain unclear, particularly how MNPs affect intercellular interactions and exacerbate the pathological process of AD. Current treatments for AD primarily rely on cholinesterase inhibitors (such as donepezil) and NMDA receptor antagonists (such as memantine), but these drugs only relieve symptoms and cannot stop disease progression, and long-term use may lead to drug resistance and adverse reactions. Therefore, exploring the key molecular mechanisms by which MNPs promote AD and developing targeted intervention strategies is of significant clinical importance. Summary of the Invention

[0003] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide the application of inhibitors targeting the COLLAGEN pathway in the preparation of drugs for treating Alzheimer's disease associated with exposure to micro- and nano-plastic particles.

[0004] The above-mentioned objective of this invention is achieved through the following technical solution:

[0005] Recent studies have revealed that extracellular matrix components, particularly collagen family proteins, play crucial roles in neuroinflammation and neurodegenerative diseases. The collagen signaling pathway, through integrin receptors (such as ITGA1 / ITGB1), mediates intercellular communication and may be involved in regulating microglia activation and neuronal damage. However, whether microneedles (MNPs) influence intercellular crosstalk by regulating the collagen pathway, thereby promoting AD progression, has not been reported. Therefore, elucidating the neuronal communication mechanisms mediated by MNPs via the collagen pathway could not only provide a new perspective for MNP neurotoxicity research but also potentially offer novel molecular targets for AD treatment.

[0006] This invention utilizes a triple co-culture system of astrocytes (SVG p12), microglia (HMC3), and neurons (SH-SY5Y). Because SH-SY5Y cells, after being induced by retinoic acid (RA) to differentiate into neuron-like cells (RA-SH-SY5Y), possess the ability to express Aβ, they have been selected as a neuron model and have become a widely accepted in vitro experimental system in Alzheimer's disease (AD) research.

[0007] Experiments revealed that MNP exposure significantly enhanced the binding of astrocyte-derived COL1A1 to the microglia surface receptors ITGA1 / ITGB1 by activating the COLLAGEN signaling pathway, thereby promoting microglia's pro-inflammatory M1 polarization (manifested as an increase in CD86 / CD206). Simultaneously, increased binding of microglia-derived COL1A2 to the neuronal surface receptors ITGA1 / ITGB1 activated the intraneuronal p38-MAPK pathway, leading to neuronal death and Aβ deposition. Knocking down COL1A1 in SVG p12 astrocytes and blocking the ITGA1 / ITGB1 receptors in HMC3 microglia significantly inhibited these pathological processes. Further research showed that MNPs, by upregulating the binding of COL1A1-ITGA1 / ITGB1 and COL1A2-ITGA1 / ITGB1 ligands, drive microglia M1 polarization and activate the neuronal p38-MAPK pathway, thus exacerbating the AD pathological process. Targeting and inhibiting the COLLAGEN pathway (e.g., by knocking down COL1A1 with siRNA to block ITGA1 / ITGB1) can reverse MNP-induced neuroinflammation and neuronal damage, improving the pathological progression of AD. This invention provides a novel approach and strategy for treating AD exacerbations caused by MNPs, and can significantly improve treatment outcomes. This invention discloses a treatment method that inhibits the COLLAGEN pathway to treat AD progression exacerbated by MNP exposure.

[0008] Therefore, the present invention provides the following new uses:

[0009] The application of inhibitors targeting the COLLAGEN signaling pathway in the preparation of drugs for treating Alzheimer's disease with increased exposure to MNPs, wherein the COLLAGEN signaling pathway is COL1A1-ITGA1 / ITGB1 (i.e., a signaling pathway composed of astrocyte-derived COL1A1 and microglia surface receptor ITGA1 / ITGB1) and / or COL1A2-ITGA1 / ITGB1 (i.e., a signaling pathway composed of microglia-derived COL1A2 and neuronal surface receptor ITGA1 / ITGB1), with type I collagen α1 (COL1A1) / type I collagen α2 (COL1A2) as ligands and integrins (ITGA1 / ITGB1) as receptors. Integrins are a large family of transmembrane cell adhesion molecules composed of non-covalent α / β heterodimers. ITGA1 / ITGB1 is a heterodimer protein composed of the integrin α1 subunit (ITGA1) and the integrin β1 subunit (ITGB1), belonging to the integrin family. They are important cell surface adhesion receptors, playing a crucial role in cell-extracellular matrix (ECM) interactions and signal transduction. Different combinations of α / β subunits determine their specific binding ligands (such as extracellular matrix components like collagen and laminin) and functions. ITGA1 (integrin α1 chain): The gene is located on chromosome 5q11.2. Its extracellular region specifically recognizes and binds to collagen (such as type I and type IV collagen) and laminin, and is a key subunit determining the ligand specificity of this heterodimer. ITGB1 (integrin β1 chain): It is one of the most widely distributed β subunits in the integrin family, and can bind to various α subunits (such as α1, α2, α5, etc.) to form different dimers. Its intracellular region can interact with intracellular cytoskeletal proteins (such as ankle proteins and peg proteins) and signaling molecules (such as FAK and Src kinases) to transmit extracellular signals into the cell and regulate cellular function.

[0010] This invention demonstrates that the pathological features of Alzheimer's disease (AD) exacerbated by MNP exposure include microglia M1 polarization (elevated CD86 / CD206 ratio), activation of the neuronal p38-MAPK pathway, neuronal loss, and Aβ deposition. The inhibitor described herein alleviates MNP-induced microglia M1 activation and neuronal loss by inhibiting the enhanced astrocyte-microglia-neuron communication mediated by the MNP-activated COLLAGEN pathway. This, in turn, alleviates the pathological features of AD exacerbated by MNP exposure, thus achieving therapeutic effects.

[0011] Furthermore, the inhibitor targeting the COLLAGEN signaling pathway is an inhibitor of the expression of ligands COL1A1 or COL1A2, or a reagent that inhibits the binding function of receptors ITGA1 / ITGB1. This thereby blocks the binding of COL1A1 and COL1A2 ligands to the ITGA1 / ITGB1 receptor.

[0012] Furthermore, the expression inhibitor is a reagent that targets and silences COL1A1 or downregulates COL1A1.

[0013] Furthermore, the reagent is siRNA of COL1A1.

[0014] Preferably, the siRNA sequence of COL1A1 is: Forward: CCAUCAAAGUCUUCUGCAA, Reverse: UUGCAGAAGACUUUGAUGG.

[0015] Preferably, the siRNA sequence of COL1A1 is: Forward: GCCAGCAGAUCGAGAACAU, Reverse: AUGUUCUCGAUCUGCUGGC.

[0016] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] This invention provides a method for treating AD progression exacerbated by MNP exposure by targeting the COLLAGEN signaling pathway. Studies in this invention show that long-term exposure to MNPs significantly aggravates cognitive impairment, neuronal loss, and Aβ deposition in AD model mice, and accelerates disease progression by promoting cellular crosstalk between astrocytes, microglia, and neurons. Further research shows that MNPs activate the COLLAGEN signaling pathway by upregulating astrocyte-derived COL1A1 and microglia-derived COL1A2, enhancing their binding to integrin receptors ITGA1 / ITGB1, thereby inducing microglia M1 polarization and neuronal p38 MAPK pathway activation, ultimately leading to neuronal death and Aβ accumulation. Interventions targeting the COLLAGEN pathway, such as siRNA silencing COL1A1, can significantly alleviate MNP-induced microglia activation and neuronal loss, improving AD pathological progression. This invention reveals for the first time the crucial role of the COLLAGEN signaling pathway in MNP-mediated AD exacerbation and provides a novel target and therapeutic strategy. Attached Figure Description

[0019] Figure 1 for si- COL1A1The COLLAGEN signaling pathway in co-cultured SVG p12 astrocytes was analyzed under PS-NP exposure. Figure 1 In Figure a, representative immunoblot images are shown in SVG p12 cells; in Figure b, quantitative analysis of COL1A1 expression in SVG p12 astrocytes is presented.

[0020] Figure 2 for si- COL1A1 Analysis of the COLLAGEN signaling pathway in HMC3 microglia exposed to PS-NPs after co-culture of SVG p12 astrocytes. Figure 2 In the middle section, a represents a representative immunoblot image in HMC3 microglia; bg represents the co-immunoprecipitation (CO-IP) analysis in HMC3 microglia: the binding of COL1A1 to ITGA (b) and ITGB (c) in HMC3 microglia; d–g represents the quantification of COL1A1 (d), COL1A2 (e), ITGA1 (f), and ITGB1 (g) input proteins in HMC3 microglia.

[0021] Figure 3 for si- COL1A1 Flow cytometry analysis of co-cultured SVG p12 astrocytes and HMC3 microglia exposed to PS-NPs. Figure 3 In Figure a, a representative flow cytometry plot shows the expression of the anti-inflammatory marker CD206 and the pro-inflammatory marker CD86; in Figure b, the quantitative analysis results are presented.

[0022] Figure 4 for si- COL1A1 Analysis of the COLLAGEN signaling pathway in RA-SH-SY5Y neurons under PS-NP exposure after processing and co-cultured SVG p12 astrocytes. Figure 4 In the figure, a is a representative immunoblot image of RA-SH-SY5Y neurons in the CO-IP experiment; b and c are CO-IP analyses of the binding of COL1A2 with ITGA1 (b) and ITGB1 (c) in RA-SH-SY5Y neurons; df is the quantitative expression of COL1A2 (d), TGA1 (e), and ITGB1 (f) in the input sample of RA-SH-SY5Y neurons.

[0023] Figure 5 For Si- COL1A1 The expression of Aβ in RA-SH-SY5Y neurons was investigated by treating co-cultured SVG p12 astrocytes and exposing them to PS-NPs. Figure 5Image a shows a representative immunoblot image of RA-SH-SY5Y neurons; image b shows the quantitative analysis of Aβ protein in RA-SH-SY5Y neurons; images c and d show the ELISA detection of Aβ protein in RA-SH-SY5Y neurons. 40 (c) and Aβ 42 (d) Concentration.

[0024] Figure 6 for si- COL1A1 Expression of the MAPK pathway in RA-SH-SY5Y neurons was investigated in co-cultured SVG p12 astrocytes after PS-NP exposure. Figure 6 In the middle section, a is a representative immunoblot image of RA-SH-SY5Y neurons; b and c are the total p38 MAPK (b) and its phosphorylation level (c) in the MAPK pathway of RA-SH-SY5Y neurons.

[0025] Figure 7 For in si- COL1A1 Cell death of RA-SH-SY5Y neurons in a ternary co-culture system was assessed under SVG p12 astrocyte and PS-NP exposure conditions. Figure 7 In the diagram, a represents a representative flow cytometry plot; b represents a quantitative analysis of cell death rate. Detailed Implementation

[0026] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0027] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0028] The cell lines used in the embodiments of this invention are: SVG p12 human astrocytes, HMC3 human microglia, and SH-SY5Y human neuroblastoma cells.

[0029] Example 1

[0030] 1. Method

[0031] S1. Cell Culture and Processing

[0032] (1) Cell Culture: We used the following human cell lines: SVG p12 astrocytes, HMC3 microglia, and SH-SY5Y neurons. All cell lines were cultured at 37°C in a humidified environment with 5% CO2. The culture medium was Dulbecco modified Eagle medium (DMEM) containing 10% fetal bovine serum and 1% penicillin-streptomycin. Mycoplasma contamination was ruled out every three months using the one-step Quickcolor mycoplasma detection kit. The cells used in the experiment were passaged 10 to 20 times to ensure the consistency of the experimental results. According to the International Committee for Certification of Cell Lines (ICLAC) database (http: / / iclac.org / databases / cross-contaminations), the cell lines used did not appear in the list of commonly misidentified cell lines.

[0033] (2) Polystyrene nanoplastics (PS-NPs) exposure experiment: To investigate the effects of PS-NPs on three types of intercellular communication, a co-culture model of the three cell types was established using 6-well plates and Transwell inserters. In the initial stage, 3.0 × 10⁶ cells were placed in a 6-well plate. 6 HMC3 microglia were seeded into the upper lumen of a Transwell culture medium in serum-free DMEM; DMEM containing 10% FBS was added to the lower lumen. After two days of culture, the microglia migrated and adhered to the underside of the dialysis membrane, and residual cells in the upper lumen were removed using sterile swabs. SH-SY5Y neurons were pretreated with 10 μM retinoic acid (RA) and cultured for 7 days, with the medium changed daily. RA-induced differentiated cells were used to model Alzheimer's disease (AD) in a ternary co-culture system. Subsequently, the Transwell cells were transferred to a pre-seeded 3.0 × 10⁶ cells / mL transwell culture medium. 6 2.5 × 10⁶ SH-SY5Y neurons were seeded in a 6-well plate. 6 SVGp12 astrocytes were cultured in DMEM medium containing 10% FBS. The SVGp12 astrocytes were divided into three groups: a reagent-free control group (Mock), a control group pre-transfected with null sequences (scramble), a PS-NPs exposure group (scramble + PS-NPs), and an intervention group pre-transfected with the Si-COL1A1 fragment to antagonize PS-NPs exposure (si-COL1A1 + PS-NPs).

[0034] After co-culturing the three cell lines for 48 hours, the cells were further treated with 0 or 50 μg / mL PS-NPs for 48 hours. In the co-culture experiment, SH-SY5Y neurons were pretreated with 10 μM retinoic acid to induce neuronal differentiation. The culture medium was changed daily for 7 days.

[0035] S2. Protein extraction, immunoprecipitation, and Western blotting

[0036] (1) Extraction of total cellular protein:

[0037] ① After processing the cells in the 6-well plate (SVG p12, HMC3, SH-SY5Y cells according to the experimental purpose), discard the old culture medium and gently wash three times with pre-cooled PBS buffer.

[0038] ② Prepare an appropriate amount of protein lysis buffer (IP lysis buffer + phosphatase / protease inhibitor) at a ratio of 100:1. After thorough mixing, add 100 µL of lysis buffer to each well and lyse on ice for 30 minutes, shaking the cell culture plate during the process.

[0039] ③ Use a pipette to transfer the cell lysate into an enzyme-free 1.5 mL EP tube.

[0040] ④ Centrifuge at 12,000 r / min and 4 ℃ for 30 minutes using a low-temperature high-speed centrifuge. The resulting supernatant is the total protein solution. Transfer it to a new 1.5 mL EP tube.

[0041] (2) Protein concentration determination:

[0042] ① Dilute Coomassie Brilliant Blue stock solution 5 times to prepare the application solution, and place it on a shaker and shake well.

[0043] ② Weigh 0.01 g of albumin and add 20 mL of deionized water to prepare a protein standard concentration of 5,000 µg / mL. Then, dilute the albumin solution by multiples to prepare eight concentration gradients (0, 78.125, 156.25, 312.5, 625, 1,250, 2,500, and 5,000 µg / mL). After mixing, add 10 µL to each well of a 96-well plate, with three replicates for each concentration.

[0044] ③ Take 2 µL of the protein sample to be tested, add 78 µL of deionized water, and mix thoroughly. Take 10 µL and add it to a 96-well plate, setting up 3 replicate wells for each protein sample. Add 200 µL of diluted Coomassie Brilliant Blue application solution, attach the membrane, and incubate at 37 ℃ for 15 minutes.

[0045] ④ Place the 96-well plate into a microplate reader and measure the OD value of each well for protein standards of various concentrations and the protein samples to be tested at a wavelength of 595 nm. Plot a standard curve with the concentration of standard albumin solution as the x-axis and the measured OD value as the y-axis. Calculate the total protein concentration of the sample based on the standard curve and the OD values ​​of the protein samples to be tested.

[0046] (3) Protein denaturation: Based on the measured protein sample concentration, add an appropriate amount of IP lysis buffer to adjust the protein sample concentration to 3 µg / µL, and add 1 volume of 5× Loading buffer to 4 volumes of protein to prepare the protein denaturation system. Vortex mix and then briefly disconnect, and denature in a metal bath at 100℃ for 10 minutes.

[0047] (4) Electrophoresis: Load 15 µL of protein sample and 5 µL of protein marker into each well. After connecting the power supply, set the voltage to 65V, change the voltage to 90V after 30 minutes, and continue electrophoresis for 1.5 hours.

[0048] (5) Transfer: After electrophoresis, remove the gel and transfer it onto the PVDF membrane. Connect the power supply and set the voltage to 100 V. The transfer time is about 1 hour.

[0049] (6) Immune response:

[0050] ① Sealing: After the transfer is completed, place the PVDF membrane in 5% skim milk powder and seal for 2 hours.

[0051] ② Primary antibody incubation: After washing the PVDF membrane three times with TBST, place the PVDF membrane in the prepared primary antibody and incubate overnight at 4 ℃ in a shaker refrigerator.

[0052] ③ Secondary antibody incubation: Place the PVDF membrane in the secondary antibody and incubate at room temperature for 1.5 hours.

[0053] (7) Strip chemiluminescence development: Under light-protected conditions, take 1 mL each of ECL luminescent solution A and solution B and mix them thoroughly. Immerse the PVDF membrane in the luminescent mixture and react for 1-2 minutes. Then place the PVDF membrane on the plate of the fluorescence image analysis system for exposure and development. The specific exposure time should be adjusted according to the experimental conditions. After exposure and development, save the strip image.

[0054] (8) Immunoprecipitation: To investigate the ligand-receptor interactions involved in changes in intercellular communication under PS-NP exposure, we performed an immunoprecipitation experiment to detect the binding of COL1A1 to ITGA1 / ITGB1 in HMC3 microglia in a cell co-culture model, and the binding of COL1A2 to ITGA1 / ITGB1 in SH-SY5Y neurons. Before the experiment, we pre-washed Protein A / G magnetic beads with IP binding buffer and then incubated them with anti-COL1A1, anti-COL1A2, or mouse IgG antibodies at room temperature for 30 minutes. 15% of the total protein lysis buffer was used as the input group sample, which was mixed with 5× loading buffer and heated at 100°C for 5 minutes. The remaining protein extracts were incubated with antibody-bound magnetic beads at room temperature with shaking for 2 hours, with isotype IgG as a negative control. After incubation, the magnetic beads were washed four times with IP washing buffer to remove non-specific bindings. The bound proteins were eluted by mixing with 5× loading buffer, heating at 100°C for 5 minutes, and analyzed by Western blotting. All protein expression levels were normalized using β-ACTIN as an internal control. For protein expression levels in Co-IP experiments, normalization to the co-precipitated protein level for each sample was performed.

[0055] S3. Plasmid Transfection: To suppress the COL1A1 ligand, we used two independent small interfering RNAs (siRNAs) targeting the COL1A1 gene (siRNA sequences are Si- COL1A1 -1: Forward: CCAUCAAAGUCUUCUGCAA, Reverse: UUGCAGAAGACUUUGAUGG; Si- COL1A1 -2: Forward: GCCAGCAGAUCGAGAACAU, Reverse: AUGUUCUCGAUCUGCUGGC). SVG p12 astrocytes were seeded in 6-well plates at a confluence of 50-70% 24 hours prior to transfection. For transfection, 100 nM siRNA was introduced into the cells using RNAFit according to the manufacturer's protocol. COL1A1 protein levels were then measured to verify inhibitory efficiency; transfected cells were used in a three-cell co-culture system when COL1A1 inhibition efficiency fell below 50%.

[0056] S4. Flow cytometry:

[0057] (1) Microglial cell polarization detection:

[0058] The treated HMC3 microglia were collected and labeled with CD206-APC and CD86-PE antibodies. Cells were incubated at 37°C in the dark for 20 minutes, then washed twice with 1 mL staining buffer, each wash performed by centrifugation at 1500 rpm for 5 minutes. After discarding the supernatant, the cell pellet was resuspended in 300 μL of staining buffer.

[0059] (2) Detection of neuronal apoptosis:

[0060] SH-SY5Y neurons were stained using the FITC Annexin V apoptosis detection kit. First, adherent and suspended neurons were washed twice with ice-cold PBS, then resuspended in the staining buffer provided in the kit. 2.5 μL of Annexin-V-FITC and 2.5 μL of propidium iodide were added, and the cells were incubated at 37°C in the dark for 15 minutes. After staining, 400 μL of diluted 1× binding buffer was added to resuspend the cells, and the mixture was gently stirred.

[0061] (3) Stream cytometry analysis and data processing

[0062] All samples were analyzed using flow cytometry. Experimental data were processed and visualized using FlowJo software.

[0063] 2. Results

[0064] like Figure 1 As shown, compared with the solvent control group (Mock), there were no significant changes in molecules in the COLLAGEN pathway in the control group (scramble), indicating that the reagents do not affect the expression of molecules in the COLLAGEN pathway. Compared with the PS-NPs exposure group (scramble+PS-NPs), 50 μg / mL of PS-NPs significantly enhanced the expression of COL1A1 in SVG p12 astrocytes, increasing ligand availability. Figure 2 The results showed enhanced binding of COL1A1 to integrins ITGA1 and ITGB1 on HMC3 microglia. This indicates that PS-NPs promote astrocyte-microglia interaction by enhancing the binding of astrocyte-derived COL1A1 to microglia's integrins ITGA1 and ITGB1. Figure 3 Flow cytometry results showed that PS-NP treatment induced the polarization of HMC3 microglia towards the pro-inflammatory M1 phenotype, manifested by an increased CD86 / CD206 ratio. These results indicate that PS-NPs drive aberrant communication between astrocytes and microglia through the COLLAGEN signaling pathway and induce microglia activation.

[0065] In downstream cell communication, PS-NPs significantly upregulated the level of COL1A2 in downstream HMC3 microglia, indicating that it is the source of this ligand. Figure 4 The results showed that COL1A2 enhanced the binding of integrins ITGA1 and ITGB1 to RA-induced SH-SY5Y neurons; Figure 5 The results show that, under this system, PS-NPs also increased the accumulation of Aβ, especially Aβ. 42 At the neuronal level, integrin activation binding to Aβ is known to trigger MAPK signaling, inducing neuronal death. Figure 6 The results showed that PS-NPs selectively activated the p38 pathway, with increased phosphorylation levels in RA-SH-SY5Y cells. Figure 7 Flow cytometry further confirmed that, in the co-culture system, PS-NP-induced p38 MAPK activation led to neuronal death.

[0066] si- COL1A1 Treatment with SVG p12 can alleviate PS-NP-induced microglial activation and neuronal death in a three-cell culture system (si-COL1A1+PS-NPs group) by inhibiting the COLLAGEN signaling pathway. This was demonstrated by CO-IP analysis, which showed that in the si- COL1A1 In the +PS-NPs group, compared with the scramble+PS-NPs group, the binding of COL1A1 derived from astrocytes to ITGA1 and ITGB1 integrins on the surface of HMC3 microglia was significantly reduced. Figure 2 b、 Figure 2 c). These results indicate that reducing COL1A1 levels in astrocytes limits its availability as a ligand for binding to HMC3 cytokines, thereby effectively inhibiting the polarization of HMC3 towards the pro-inflammatory M1 phenotype under PS-NP treatment, which is verified by the decrease in the CD86 / CD206 ratio by flow cytometry. Figure 3 ).

[0067] Further investigation was conducted into downstream signal transduction between microglia and neurons in the co-culture system. Compared to the exposed group (scramble + PS-NPs), si-COL1A1 transfection of SVG p12 cells resulted in reduced COL1A2 expression in HMC3 microglia. Figure 2 e). COL1A2 binds less to ITGA1 and ITGB1 integrins on RA-induced SH-SY5Y neurons ( Figure 4 b、 Figure 4 c). COL1A2 expression also decreased synchronously in neurons, suggesting that ligand transfer from microglia to neurons was impaired. Figure 4d). Notably, even under COL1A1 silencing conditions, Aβ accumulation persists, especially Aβ... 42 ( Figure 5 ), but the activation level of p38 MAPK ( Figure 6 ) and a significant reduction in the degree of neuronal death ( Figure 7 This suggests that Aβ alone is insufficient to induce neurotoxicity under PS-NP exposure. The COLLAGEN signaling pathway plays a crucial role in amplifying Aβ toxicity.

[0068] This invention reveals for the first time the key role of the COLLAGEN signaling pathway in MNP-mediated AD exacerbation and discloses a treatment method that inhibits the COLLAGEN pathway to treat AD progression exacerbated by MNP exposure, providing a novel target and treatment strategy for treating AD exacerbation caused by MNPs.

Claims

1. The application of inhibitors targeting the COLLAGEN pathway in the preparation of drugs for treating Alzheimer's disease associated with exposure to micro / nanoplastics particles, characterized in that, The COLLAGEN pathway is COL1A1-ITGA1 / ITGB1 and / or COL1A2-ITGA1 / ITGB1, wherein COL1A1 and COL1A2 are ligands and ITGA1 / ITGB1 are receptors; the inhibitor is a COL1A1 siRNA; the COL1A1 siRNA sequence is Forward: CCAUCAAAGUCUUCUGCAA, Reverse: UUGCAGAAGACUUUGAUGG or Forward: GCCAGCAGAUCGAGAACAU, Reverse: AUGUUCUCGAUCUGCUGGC.

2. The application according to claim 1, characterized in that, The drug alleviates microglia M1 activation and neuronal loss by inhibiting enhanced astrocyte-microglia-neuron communication mediated by the COLLAGEN pathway activated by micro- and nano-plastic particles, thereby improving the pathological progression of Alzheimer's disease exacerbated by micro- and nano-plastic particle exposure.

3. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable excipients.

Images