Application of targeting inhibition of MSR1-PI3K / AKT signaling pathway in treatment of renal injury caused by polylactic acid microplastic particles

Abstract

This invention discloses the application of targeting and inhibiting the MSR1-PI3K / AKT signaling pathway in the treatment of kidney injury induced by polylactic acid (PLA) microplastic particles (PLMPs). The study shows that oligomeric PLA MPs recognize and activate the downstream PI3K / AKT signaling pathway via MSR1, significantly upregulating the expression of the chemokine CCL2, thereby amplifying the macrophage-mediated inflammatory cascade. By inhibiting MSR1 or blocking the PI3K / AKT signaling pathway, the macrophage accumulation, inflammation activation, and renal tissue structural damage induced by oligomeric PLA MPs can be significantly reduced, thereby treating PLA MPs-induced kidney injury. This invention reveals for the first time the key regulatory role of the MSR1-PI3K / AKT signaling axis in PLA MPs-induced kidney injury, providing a new molecular target and intervention strategy for the prevention and treatment of degradable MPs-related kidney diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and more specifically, relates to the application of targeted inhibition of the MSR1-PI3K / AKT signaling pathway in the treatment of kidney injury caused by polylactic acid microplastic particles. Background Technology

[0002] Polylactic acid (PLA), a biodegradable plastic widely used in medical and food packaging, breaks down during production, use, and natural degradation, generating microplastics (MPs), which have become an increasingly prominent emerging pollutant in the environment. Unlike traditional non-degradable plastics such as polyethylene (PE) and polystyrene (PS), PLA degrades in vitro and in vivo to form low-molecular-weight oligomers, PLA MPs. These products possess unique physicochemical properties and biological behaviors, exhibiting stronger potential toxicity. Existing studies have shown that low-molecular-weight PLA oligomers can induce significant toxic reactions in the liver and nervous system, but their accumulation characteristics and toxic mechanisms in the kidneys, a vital excretory organ, lack systematic research.

[0003] As the body's primary organ for filtration and excretion, the kidneys are particularly sensitive to the accumulation and toxic effects of polymorphonuclear leukocytes (PLA MPs). Recent research has revealed a significant molecular weight dependence in the accumulation of PLA MPs in the kidneys: among all organs tested, the kidneys showed a significantly higher accumulation of low-molecular-weight oligomeric PLA MPs than high-molecular-weight polymeric PLA MPs. This phenomenon suggests a unique interaction between PLA MP degradation products and kidney tissue, and that low-molecular-weight oligomeric PLA MPs may acquire selective enrichment in the kidneys through specific molecular mechanisms. However, the specific mechanism of this molecular weight-dependent enrichment remains unclear.

[0004] Macrophage scavenger receptor 1, also known as MSR1, is a protein encoded by the MSR1 gene. MSR1 is also designated CD204 (differentiation cluster 204). This gene encodes class A macrophage scavenger receptors, including three distinct types (1, 2, and 3), produced by alternative splicing of this gene. These receptors, or isoforms, are trimeric whole membrane glycoproteins that have been involved in many macrophage-related physiological and pathological processes, including atherosclerosis, Alzheimer's disease, and host defense. Currently, the role of macrophage scavenger receptor 1 in the renal enrichment of PLA MPs has not been reported. Summary of the Invention

[0005] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide the application of an inhibitor that targets and inhibits the MSR1-PI3K / AKT signaling pathway in the preparation of a medicament for treating kidney damage caused by polylactic acid microplastic particles.

[0006] The above-mentioned objective of this invention is achieved through the following technical solution: This invention experimentally revealed that at the cellular level, exposure to oligomeric PLA MPs (low molecular weight PLA MPs) significantly upregulated the expression of the chemokine CCL2 in macrophages (RAW264.7) and enhanced macrophage migration. Further, MSR1-specific siRNA was used to knock down CCL2 expression in macrophages. Msr1 Expression of this substance can significantly inhibit the upregulation of CCL2 and pro-inflammatory cytokines induced by oligomeric PLA MPs. Il-1β and Tnf-α The expression of MSR1 indicates that it is a key receptor mediating macrophage recruitment and inflammatory response induced by oligomeric PLA MPs. Based on this, downstream inflammatory pathways were screened, revealing significant activation of the PI3K-AKT pathway; targeting and inhibiting this pathway effectively reversed oligomeric PLA MPs-induced macrophage recruitment and inflammatory cytokine release, confirming the core mediating role of the PI3K-AKT pathway in this process. Further mechanistic validation showed that siRNA knockdown... Msr1 Subsequently, AKT phosphorylation levels significantly decreased, thus confirming MSR1's role as an upstream regulatory molecule in the PI3K-AKT pathway. Specifically, MSR1 can mediate macrophage recruitment and activation by recognizing oligomeric PLA MPs and activating the downstream PI3K / AKT signaling pathway, thereby triggering local renal inflammatory responses and tissue damage. This provides a novel approach and strategy for treating inflammatory damage caused by oligomeric PLA MPs exposure and can significantly improve treatment outcomes.

[0007] This invention continues to utilize animal models, establishing a mouse model of kidney injury by continuous gavage administration of oligomeric PLA MPs (25 mg / kg). Intervention was then conducted using the MSR1 inhibitor fucoidan and the PI3K / AKT specific inhibitor LY294002. The protective effect of targeting the MSR1-PI3K / AKT pathway on kidney injury was systematically evaluated. The study found that using MSR1 or PI3K / AKT signaling pathway inhibitors effectively alleviated macrophage activation, upregulation of cytokines and chemokines, and renal tissue structural damage induced by oligomeric PLA MPs. These results indicate that the MSR1-PI3K / AKT pathway plays a crucial role in MNP-induced kidney injury, and targeting this pathway can significantly reduce microplastic-induced nephrotoxicity.

[0008] This invention demonstrates that exposure to oligomeric PLA MPs significantly upregulates the PI3K / AKT signaling pathway by activating the MSR1 receptor, inducing macrophage infiltration and inflammatory responses, and exacerbating renal tissue structural damage through molecular mechanisms. Further experimental results show that using MSR1 inhibitors or PI3K / AKT inhibitors can significantly alleviate renal damage and inflammatory responses induced by oligomeric PLA MPs, improve glomerular damage, reduce macrophage infiltration, and decrease PLA MP accumulation, thereby treating PLA MPs-induced renal injury.

[0009] Therefore, the present invention provides the following new uses: Application of agents targeting and inhibiting the MSR1-PI3K / AKT signaling pathway in the preparation of drugs for treating kidney injury caused by polylactic acid (PLA) microplastic particles. Since PLA forms low-molecular-weight oligomers (PLA MPs) during its degradation in vivo and in vitro, this study provides an opportunity to develop agents targeting and inhibiting the MSR1-PI3K / AKT signaling pathway for treating kidney injury caused by oligomeric PLA MPs. Specifically, kidney injury induced by exposure to oligomeric PLA MPs is characterized by increased macrophage infiltration (manifested as an increase in CD68-positive cells), upregulation of pro-inflammatory cytokines IL-1β and chemokine CCL2, damage to kidney tissue structure, and abnormal deposition of oligomeric PLA MPs in kidney tissue. The drug described herein treats polylactic acid (PLA) microplastic particle-induced kidney injury and inflammatory response by inhibiting macrophage activation and the inflammatory cascade mediated by the MSR1-PI3K / AKT signaling pathway, thereby improving glomerular damage, reducing macrophage infiltration, and decreasing PLA microplastic particle accumulation.

[0010] Furthermore, the reagent that targets and inhibits the MSR1-PI3K / AKT signaling pathway is an MSR1 inhibitor or a PI3K / AKT pathway inhibitor.

[0011] Furthermore, the MSR1 inhibitor is a reagent that targets and inhibits the function of the MSR1 receptor, and the PI3K / AKT pathway inhibitor is a reagent that targets and inhibits the activation of the PI3K / AKT signaling pathway.

[0012] Furthermore, the reagent that targets and inhibits the function of the MSR1 receptor is an interfering RNA, antibody, peptide, small molecule compound, or biological macromolecule that targets and inhibits the MSR1 receptor.

[0013] Preferably, the interfering RNA is siRNA.

[0014] Preferably, the siRNA sequence is si- Msr1-1: Forward: GAAUGUCAGAGUCCGUGAA, Reverse: UUCACGGACUCUGACAUUC; or si- Msr1 -2: Forward: CGACCUUAUAGACACGGAA, Reverse: UUCCGUGUCUAUAAGGUCG.

[0015] Preferably, the biomolecule is fucoidan, CAS Registry No. 9072-19-9.

[0016] Furthermore, the reagent that targets and inhibits the activation of the PI3K / AKT signaling pathway is the PI3K / AKT specific inhibitor LY294002, CAS Registry Number 154447-36-6.

[0017] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0018] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a novel method for treating kidney injury induced by oligomeric PLA MPs exposure by targeting and inhibiting the MSR1-PI3K / AKT signaling pathway. Studies in this invention show that oligomeric PLA MPs exposure significantly exacerbates kidney injury in mice, primarily manifested as increased macrophage infiltration and enhanced local renal inflammation. Mechanistic studies indicate that oligomeric PLAMPs activate the MSR1 receptor, thereby upregulating the PI3K / AKT signaling pathway, promoting the production of pro-inflammatory cytokines, and ultimately leading to renal tissue structural damage. Further analysis reveals that MSR1 regulates macrophage recruitment and activation through the PI3K / AKT signaling axis, mediating the inflammatory cascade and exacerbating the kidney injury process. Targeted interventions, such as MSR1 inhibitors or PI3K / AKT pathway inhibitors, can significantly inhibit the activation of this signaling axis, reduce macrophage infiltration and inflammatory response, and improve renal pathological damage. This invention reveals for the first time the key regulatory role of the MSR1-PI3K / AKT signaling pathway in kidney injury induced by oligomeric PLA MPs, providing new intervention targets and treatment strategies, and laying a solid theoretical and practical foundation for addressing kidney diseases caused by environmental pollution related to biodegradable microplastics. Attached Figure Description

[0019] Figure 1 This study aimed to validate the role of the MSR1-PI3K / AKT pathway in the in vitro response of macrophages induced by oligomeric PLA MPs. Figure 1Image A shows the CCL2 content in the cell supernatant after PLA MPs treatment of RAW264.7 cells, as detected by an ELISA kit; Image B shows a representative image of macrophage migration after treatment with the CCR2-specific inhibitor INCB3284; Image C shows the quantitative results of the number of migrating cells; Image D shows the knockdown... Msr1 The relative content of CCL2 in the supernatant of RAW264.7 cells after treatment; E-F represent the concentrations of PLA multimers (PLA MPs) and oligomers (PLA MPs). Il-1β (E) and Tnf-α (F) relative expression level; G~H are knockdown Msr1 Post-RAW264.7 cells Il-1β (G) and Tnf-α (H) represents the relative expression level; I represents the heatmap of PI3K / AKT pathway-related gene expression in the transcriptome; J represents the Western blot analysis of p-AKT / AKT protein expression and quantification; K-N represent the AKT phosphorylation level (K) in RAW264.7 cells after treatment with LY294002 inhibitor. Il-1β (L) Tnf-α Relative expression levels of (M) and CCL2 (N); O–P represent representative bands (O) and quantitative results (P) of p-AKT / AKT after Msr1 knockdown. Each group n =3.

[0020] Figure 2 To investigate the effect of the MSR1-PI3K / AKT pathway on renal injury induced by oligomeric PLA MPs. Figure 1 Figure A shows the experimental design of the intervention with fucoidan and LY294002; B-C show the representative immunoblot bands and relative protein levels of MSR1 protein after inhibitor intervention; D-E show the representative immunoblot bands and relative protein levels of p-AKT / AKT after inhibitor intervention; F shows the quantitative results of glomerular injury rate after intervention; G shows the relative quantitative results of CD68 positive cells after intervention; H shows the relative quantitative results of PLA number after intervention; I shows the relative quantitative results of PLA number in macrophages after intervention; J shows the relative quantitative results of IL-1β fluorescence intensity in mouse kidneys after intervention; K shows the relative quantitative results of CCL2 fluorescence intensity in mouse kidneys after intervention; L shows the HE staining of mouse kidneys after intervention (black arrows indicate renal inflammatory infiltration) and CD68... + Representative images of immunofluorescence, PLA autofluorescence (orange arrows indicate PLA MPs phagocytosed by macrophages; red arrows indicate unphagocytosed PLA MPs), IL-1β, and CCL2 expression. Each group n =6 mice. Detailed Implementation

[0021] 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.

[0022] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0023] This invention comprises two parts: in vitro experiments and in vivo experiments. The cell line used is RAW264.7 cells, and the animal model used is male mice of the C57BL / 6J strain.

[0024] Example 1: Effect of inhibiting the MSR1-PI3K / AKT pathway on PLA oligomer-induced kidney injury 1. Method S1. Cell Culture and Processing (1) Cell Culture: We used the following mouse cell line: mouse mononuclear macrophage leukemia cell line (RAW264.7 cells). 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.

[0025] (2) PLA MPs exposure assay: To investigate the effects of PLA MPs exposure on RAW264.7 cells, RAW264.7 cells were cultured at 3 × 10⁶ cells per well. 5 Cells were seeded into 6-well plates and incubated overnight, followed by exposure to PLAMPs (10 or 40 μg / mL) in different polymeric states for 24 hours. Control wells received no microparticle treatment.

[0026] (3) Transwell migration assay: RAW264.7 cells were transported at a rate of 1×10⁻⁶ cells / year. 5 3 × 10⁶ cells / well are inoculated in the upper chamber of the Transwell chamber, and 3 × 10⁶ cells / well are inoculated in the lower chamber. 5RAW264.7 cells per well were co-cultured in DMEM complete medium for 24 h to allow them to adhere. Subsequently, only the lower chamber cells were treated with oligomeric PLA MPs (treatment time 24 h), with some experimental groups also receiving combined intervention with the chemokine receptor 2 (CCR2) inhibitor INCB3284 (three replicates per group). Upper chamber cells were not treated. After treatment, cells that migrated to the lower membrane surface were fixed and stained with crystal violet.

[0027] (4) Transient transfection with siRNA: RAW264.7 cells were transfected at a rate of 3 × 10⁻⁶ mcg / mL. 5 Cells were seeded at a density of 10 cells / well in six-well plates and cultured in complete medium at 37°C and 5% CO2 for 24 h. Transfection was performed when cell confluence reached 50%–60%. 50 μM siRNA (si-) stored at -20°C was used. Cd36 -1: Forward: GGAUCUGAAAUCGACCUUA (SEQ ID No.1), Reverse: UAAGGUCGAUUUCAGAUCC (SEQ ID No.2); si -Cd36 -2: Forward: GCACCACUGUGUACAGACA (SEQ ID No.3), Reverse: UGUCUGUACACAGUGGUGC (SEQ ID No.4); si- Msr1 -1: Forward: GAAUGUCAGAGUCCGUGAA (SEQ ID No. 5), Reverse: UUCACGGACUCUGACAUUC (SEQ ID No. 6); si- Msr1-2: Forward: CGACCUUAUAGACACGGAA (SEQ ID No. 7), Reverse: UUCCGUGUCUAUAAGGUCG (SEQ ID No. 8) and negative control siNC (Negative Control: Forward: UUCUCCGAACGUGUCACGU (SEQ ID No. 9), Reverse: ACGUGACACGUUCGGAGAA (SEQ ID No. 10)) were diluted to a working concentration of 50 nM according to experimental requirements. Simultaneously, the transfection reagent was diluted to the corresponding working concentration. The diluted siRNA and transfection reagent were mixed in an enzyme-free EP tube, gently pipetted to mix, and incubated at room temperature for 20 min. The mixture was then gently added to complete culture medium and mixed, then transferred to a six-well plate, gently shaken to mix, and incubated for another 24 h for subsequent experiments.

[0028] S2. Animal Model (1) Animal housing: This study strictly followed the "Regulations on the Management of Laboratory Animals" and the 3R principle (replace, reduce, optimize) to minimize the number of laboratory animals used and reduce their suffering while ensuring animal welfare. Eight-week-old male C57BL / 6J mice weighing 18–22 g were selected. All animals were housed in an SPF environment, with each cage equipped with a sterile filter cover, maintaining a temperature of 23–25°C and humidity of 50–60%, and a 12-hour light-dark cycle (light time 8:00–20:00). Mice had free access to food and water.

[0029] (2) Oligomeric PLA MPs exposure experiment: such as Figure 2 As shown in Figure A, a 28-day repeated gavage experiment was conducted using 36 male C57BL / 6J mice to investigate the biodistribution and renal damage of oligomeric PLA MPs in mice under the intervention of fucoidan sulfate (an MSR1 inhibitor) and LY294002 (a PI3K / AKT signaling pathway inhibitor). Both inhibitors were administered via intraperitoneal injection. Mice were randomly divided into six groups (each group...). n =6): Control group, oligomeric PLAMPs group (25 mg / kg), Fucoidan group (100 μg / animal, administered every other day), oligomeric PLAMPs+Fucoidan group, LY294002 group (50 mg / kg, three times a week), and oligomeric PLAMPs+LY294002 group. Animals were euthanized by cervical dislocation on day 28 of the experiment.

[0030] S3. Protein extraction and Western blotting (1) Extraction of total protein: ① Kidney Sample Collection: Total Kidney Protein Extraction: After each experimental cycle, mice were anesthetized by intraperitoneal injection of 3% sodium pentobarbital solution (2 mL / kg·BW). After complete anesthesia, the mice were fixed supine on a dissecting board, and the ribs on both sides were cut along the sternum. The anterior thoracic wall was everted and fixed with hemostatic forceps to fully expose the heart. Then, the injection needle was inserted into the left ventricle of the mouse, and pre-cooled physiological saline was continuously perfused until the effluent from the right atrium was completely clear. Kidney tissue used for histological examination was first perfused with pre-cooled 4% paraformaldehyde (PFA) (approximately 20 mL), and after complete dissection, it was transferred to 4% PFA solution for pre-fixation for 24 h in preparation for subsequent paraffin embedding. Kidney tissue used for molecular experiments was directly dissected for experiments or stored at -80℃ after perfusion with physiological saline.

[0031] ② Kidney tissue: 200 mg of mouse kidney tissue was placed in a 2 mL EP tube. A suitable amount of protein lysis buffer was prepared at a ratio of 100:1, and 400 μL of protein lysis buffer (RIPA lysis buffer + phosphatase / protease inhibitor) was added. Magnetic beads were then added, and homogenization was performed using a high-speed, low-temperature tissue homogenizer. After homogenization, the mixture was placed on ice for 30 minutes. Next, the mixture was centrifuged at 13,000 g at 4°C for 30 minutes, and the supernatant was collected as the total protein extract. This supernatant was transferred to a new 1.5 mL EP tube. Cell samples: 100 μL of protein lysis buffer was added to each well of a six-well plate, and the mixture was lysed on ice for 30 minutes. The lysis buffer was then collected into a 1.5 mL EP tube and incubated on ice for another 30 minutes. The mixture was then centrifuged at 4°C and 13,000 rpm for 30 minutes, and the supernatant was collected for later use.

[0032] (2) Protein concentration determination: ① Prepare an albumin standard solution with a concentration of 1 mg / mL, and dilute it equally to make 7 gradient standard solutions with different concentrations.

[0033] ② Take 2 μL of each protein sample and dilute it 30-fold to 60 μL with ultrapure water. Add the diluted protein sample and standard solution to a 96-well plate, with two replicates for each sample, and add 20 μL of diluted sample to each well.

[0034] ③ Add 200 μL of the prepared BCA working solution to each well. Seal the 96-well plate with a sealing film and incubate it in an oven at 37°C for 30 minutes.

[0035] 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 562 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.

[0036] (3) Protein denaturation: Based on the calculated protein concentration, the original protein sample was prepared into a protein sample with a concentration of 4 Hg / uL using protein lysis buffer. 5× loading buffer was added to the protein sample at a ratio of 4:1 between the protein sample volume and the loading buffer volume. After vortexing and mixing, the sample was briefly agitated and denatured in a metal bath at 100℃ for 10 min. The sample was then stored at -20℃ for later use.

[0037] (4) Glue preparation: After cleaning the glass plate with deionized water, place it in an oven to dry. After drying, place the long plate on the outside and the short plate on the inside, ensuring the bottom surfaces are flush. Secure and seal the bottom of the glass plate with wedges. After checking that both sides are clamped tightly, begin glue pouring. After thoroughly mixing the separating glue containing 8% acrylamide, use a pipette to pour it into the glass plate to the designated position. Use isopropanol liquid sealant to promote the polymerization of the separating glue. Let it stand at room temperature for 40 minutes (the specific solidification time depends on the room temperature) to allow the separating glue to fully solidify. After the separating glue has solidified, discard the isopropanol and rinse with ultrapure water. Then, pour the freshly prepared 4% acrylamide concentrate into the glass plate, carefully insert a comb to remove air bubbles from the edges, secure with clamps, and let it stand at room temperature for 40 minutes. Allow the concentrate to fully solidify.

[0038] (5) Electrophoresis: Prepare the electrophoresis tank and pour electrophoresis buffer into the tank to the specified mark. After the stacking gel solidifies, slowly pull out the comb vertically upwards, add 8 μL of protein sample to each well, and add 5 μL of protein marker to the wells at both ends. After connecting the power supply, set the voltage to 60V, and after 30 minutes, change the voltage to 95V and continue electrophoresis for 1.5 hours. Select the termination time according to the molecular weight of the target band.

[0039] (6) Transfer: Remove the stacking gel from the gel and preheat the PVDF membrane (8.5x5cm) by soaking it in methanol for 1 minute. Carefully assemble the transfer sandwich structure in the following order: white side of the transfer clamp, black sponge pad, thick filter paper, PVDF membrane, gel, thick filter paper, black sponge pad, and black side of the transfer clamp. Insert the assembled transfer clamp into the electrophoresis tank, pour in the transfer buffer, add an ice pack to the electrophoresis tank, and turn on the power. Transfer the membrane at 100V for 2 hours.

[0040] (7) Immune response: ① Blocking: After the transfer is complete, place the PVDF membrane in a 5% skim milk powder solution, shake well, and block at room temperature for 2 hours. After blocking, wash three times with TBST for 10 minutes each time.

[0041] 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.

[0042] ③ Secondary antibody incubation: Place the PVDF membrane in the secondary antibody and incubate at room temperature for 1 hour.

[0043] (8) 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.

[0044] (9) Image analysis: ImageJ software was used to perform quantitative analysis of the images and calculate the relative expression levels of proteins.

[0045] 2. Results (1) Results of in vitro cell experiments are as follows Figure 1 As shown, the effect of PLA MPs on the secretion and migration of the macrophage chemokine CCL2 was first examined. ELISA results showed that the CCL2 content in the cell supernatant of the oligomeric PLA MPs treatment group was significantly higher than that of the control group and the multimeric PLA MPs group. Figure 1 (A). Transwell migration assays showed that the migration ability of macrophages induced by oligomeric PLA MPs was significantly inhibited after using the CCR2-specific inhibitor INCB3284, and the number of migrating cells decreased in a dose-dependent manner. Figure 1 (B-C) Further investigation into the regulatory role of MSR1 is needed. Knockdown Msr1 Following gene administration, oligomeric PLAMPs induced a significant decrease in CCL2 secretion. Figure 1 (D). Simultaneously, detection of inflammatory factor expression revealed that oligomer PLA MPs could be significantly upregulated. Il-1β and Tnf-α mRNA levels, while the effect of multimer PLA MPs is weaker ( Figure 1 (E~F); knockdown Msr1 Subsequently, oligomeric PLA MPs induced Il-1β and Tnf-α The upregulation was significantly suppressed. Figure 1 Transcriptome analysis showed that the expression of genes related to the PI3K / AKT signaling pathway was significantly upregulated in the oligomeric PLA MPs treatment group (G-H). Figure 1Western blot analysis showed that oligomeric PLA MPs significantly increased AKT phosphorylation levels, while polymeric PLA MPs had no significant activating effect. Figure 1 (J). The PI3K / AKT inhibitor LY294002 can effectively block AKT phosphorylation induced by oligomeric PLA MPs ( Figure 1 (Middle K), and significantly reversed Il- 1β and Tnf-α upregulation of mRNA and CCL2 secretion ( Figure 1 (Middle L~N). Finally, knock down. Msr1 It can significantly inhibit AKT phosphorylation induced by oligomeric PLA MPs, while knocking down Cd36 This function is not available. Figure 1 (O to P). The above results indicate that oligomeric PLA MPs activate the PI3K / AKT signaling pathway through MSR1, thereby promoting CCL2 secretion and inflammatory factor expression, driving macrophage migration and amplifying the inflammatory response.

[0046] (2) Results of in vivo animal experiments as follows Figure 2 As shown, the immunoblotting results indicated that, compared with the group treated with oligomeric PLA MPs alone, intervention with Fucoidan but not LY294002 significantly downregulated the expression level of MSR1 protein in kidney tissue. Figure 2 Both (B-C) and (C) significantly inhibit AKT phosphorylation. Figure 2 (D-E) confirmed that the two inhibitors effectively blocked the activation of the MSR1-PI3K / AKT signaling pathway; quantitative statistics of glomerular injury rate showed that glomerular structural damage was significantly reduced after inhibitor intervention ( Figure 2 The relative quantitative results of CD68-positive macrophage numbers showed that inhibitor intervention significantly reduced macrophage infiltration induced by oligomer PLA MPs (in the middle F); Figure 2 The relative quantitative analysis of total oligomeric PLA MPs and intracellular PLA content in macrophages showed that inhibitory intervention effectively reduced the accumulation of oligomeric PLA MPs in kidney tissue and macrophages. Figure 2 The relative quantitative results of IL-1β and CCL2 fluorescence intensity in mouse kidneys showed that inhibitor intervention significantly suppressed the expression of pro-inflammatory factors and chemokines induced by oligomeric PLAMPs. Figure 2 J-K); HE staining of mouse kidneys after intervention (black arrows indicate inflammatory infiltration areas), CD68 +Representative images of immunofluorescence (green), PLA autofluorescence (orange arrows indicate PLAMPs phagocytosed by macrophages; red arrows indicate unphagocytosed PLA MPs), IL-1β, and CCL2 immunofluorescence staining, visually demonstrating that inhibitor intervention can effectively alleviate renal inflammatory pathological changes, reduce PLA accumulation, and inhibit the expression of inflammatory factors. Figure 2 (L). The above results indicate that targeting and inhibiting the MSR1-PI3K / AKT signaling pathway can effectively alleviate kidney injury induced by oligomer PLA MPs.

Claims

1. Application of reagents targeting and inhibiting the MSR1-PI3K / AKT signaling pathway in the preparation of drugs for treating kidney damage caused by polylactic acid microplastic particles.

2. The application according to claim 1, characterized in that, The drug treats polylactic acid (PLA) microplastic particle-induced kidney damage by inhibiting macrophage activation and inflammatory cascade mediated by the MSR1-PI3K / AKT signaling pathway, reducing glomerular damage, decreasing macrophage infiltration, and reducing PLA microplastic particle accumulation.

3. The application according to claim 1, characterized in that, The reagents that target and inhibit the MSR1-PI3K / AKT signaling pathway are MSR1 inhibitors or PI3K / AKT pathway inhibitors.

4. The application according to claim 3, characterized in that, The MSR1 inhibitor is a reagent that targets and inhibits the function of the MSR1 receptor, and the PI3K / AKT pathway inhibitor is a reagent that targets and inhibits the activation of the PI3K / AKT signaling pathway.

5. The application according to claim 4, characterized in that, The reagent that targets and inhibits the function of the MSR1 receptor is an interfering RNA, antibody, peptide, small molecule compound, or biological macromolecule that targets and inhibits the MSR1 receptor.

6. The application according to claim 5, characterized in that, The interfering RNA is siRNA.

7. The application according to claim 6, characterized in that, The siRNA sequence is si- Msr1 -1: Forward: GAAUGUCAGAGUCCGUGAA, Reverse: UUCACGGACUCUGACAUUC; or si- Msr1 -2: Forward: CGACCUUAUAGACACGGAA, Reverse: UUCCGUGUCUAUAAGGUCG.

8. The application according to claim 5, characterized in that, The biomolecule in question is a brown algae polysaccharide sulfate.

9. The application according to claim 4, characterized in that, The reagent that targets and inhibits the activation of the PI3K / AKT signaling pathway is the PI3K / AKT specific inhibitor LY294002.

10. The application according to any one of claims 1 to 9, characterized in that, The drug also includes pharmaceutically acceptable excipients.

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