Application of Piezo1 inhibitor in preparation of medicine for treating keratoconus

Abstract

The invention relates to application of a Piezo1 inhibitor in preparation of a medicine for treating keratoconus, and belongs to the technical field of preparation and application of biological medicines. By inhibiting a Piezo1 signal channel, overexpression of matrix metalloproteinase (MMPs) and release of inflammatory factors are reduced, so that thinning of corneal basis and disease progression are delayed or prevented. Gene silencing, pharmacological intervention, animal model construction and multi-dimensional detection verification prove that the GsMTx4 eye drops have good safety, effectiveness and conversion application prospects, and a new strategy and an experimental basis are provided for medicine treatment of cornea conus.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical preparation and application technology, specifically relating to the application of a Piezo1 inhibitor in the re-preparation of keratoconus drugs. Technical Background

[0002] Keratoconus (KC) is a common, progressive corneal disease characterized by thinning and forward bulging of the corneal stroma, leading to irregular astigmatism and visual impairment. It typically begins in adolescence, progresses rapidly, and has a poor prognosis, imposing a heavy economic and social burden on patients and society.

[0003] In recent years, numerous studies have suggested that keratoconus is not merely a simple corneal biomechanical abnormality, but is also closely related to chronic inflammatory responses. Upregulation of inflammatory factors, such as interleukins (IL-1β, IL-6), tumor necrosis factor-α (TNF-α), and matrix metalloproteinases (MMPs), has been detected in patients' tears and corneal tissue. Among these, abnormally high expression of MMPs is considered a significant cause of corneal collagen degradation and stromal thinning. However, the upstream signaling mechanisms triggering these inflammations and tissue degradation remain highly controversial.

[0004] Although the exact pathogenesis of keratoconus is not fully understood, the abnormal over-release of matrix metalloproteinases (MMPs) is widely recognized as a core factor leading to corneal collagen degradation and progressive thinning of the cornea. However, the upstream causes of MMP overexpression remain unclear. Previous studies suggest that mechanical factors (such as prolonged eye rubbing) are important risk factors. Piezo1, as a classic mechanosensitive ion channel, can be activated under mechanical stimulation and mediates downstream signaling responses, inducing excessive MMP secretion, providing a potential molecular mechanism for understanding the occurrence and progression of keratoconus.

[0005] Clinically, due to the unclear pathogenesis, keratoconus lacks effective medical treatments or drug interventions, and patients cannot slow or stop disease progression through systemic or topical medications. Current treatments mainly rely on surgical interventions, including corneal cross-linking and corneal transplantation. However, these methods have significant limitations: corneal cross-linking has unstable efficacy, requiring repeated surgeries for some patients; corneal transplantation faces challenges such as donor shortages, high surgical risks, and postoperative rejection.

[0006] Therefore, developing a drug treatment strategy targeting key molecular mechanisms has become an urgent need to improve patients' long-term visual function outcomes. Summary of the Invention

[0007] The purpose of this invention is to provide an application of Piezo1 inhibitor in the preparation of keratoconus drugs, in order to solve the key problem that existing treatments cannot target and inhibit corneal stroma degradation and inflammatory response, achieve effective treatment of keratoconus and provide a related research platform and drug direction.

[0008] On one hand, the present invention provides the use of a Piezo1 inhibitor in the preparation of a drug for treating keratoconus.

[0009] Preferably, the piezo1 inhibitor is GsMTx4.

[0010] On the other hand, the present invention also provides a drug for treating keratoconus, employing the following technical solution: A medicine for treating keratoconus, said medicine comprising a pharmacologically effective concentration of GsMTx4.

[0011] Preferably, the drug can inhibit the release of matrix metalloproteinases in corneal stromal cells and the activation of the NLRP3 inflammasome, thereby reducing the secretion of inflammatory factors.

[0012] Preferably, the drug is an eye drop.

[0013] Preferably, the concentration of GsMTx4 in the eye drops is 1-10 μM.

[0014] Preferably, the concentration of GsMTx4 in the eye drops is 5 μM.

[0015] In summary, the beneficial effects of the present invention are as follows: 1. In this invention, the specific inhibitor GsMTx4 is used to effectively inhibit the excessive release of MMPs from corneal stromal cells and the secretion of inflammatory factors such as IL-1β and IL-18. Furthermore, when GsMTx4 is formulated into an eye drop at a concentration of 5 μM, it can completely prevent corneal thinning in a mouse model without cytotoxicity or adverse ocular reactions. It balances safety and efficacy, filling the clinical gap of lacking effective medical drugs for keratoconus.

[0016] 2. In this invention, GsMTx4 inhibits the activity of Piezo1 protein through pharmacological means, while Piezo1 gene silencing siRNA blocks the synthesis of Piezo1 protein at the gene level. The two work together to block the Piezo1 signaling pathway from two directions: inhibiting activity and reducing synthesis, thereby more thoroughly inhibiting the excessive release of matrix metalloproteinases and the activation of inflammasomes, and enhancing the therapeutic effect on keratoconus. Attached Figure Description

[0017] Figure 1This is a comparison of the relative mRNA expression levels of MMPs in corneal stromal cells of patients with keratoconus and healthy controls in Experiment Example 1 of this invention. Figure 2 Figure A shows the Western blot results of Piezo1, NLRP3, ASC, and IL-1β p17 protein expression in corneal tissues of keratoconus patients and healthy controls in Experiment Example 1 of this invention. Figure 2 Figure B is a comparison of the relative expression levels of Piezo1, ASC, NLRP3, and IL-1β proteins in the corneal tissues of keratoconus patients and healthy controls in Experiment Example 1 of this invention. Figure 2 Figure C shows the Western blot results (repeated verification) of the expression of Piezo1, NLRP3, ASC, and IL-1βp17 proteins in the corneal tissues of keratoconus patients and healthy controls in Experiment Example 1 of this invention. Figure 2 Figure D is a comparison of the relative expression levels of Piezo1, ASC, IL-1β, and NLRP3 proteins in corneal tissues of keratoconus patients and healthy controls in Experimental Example 1 of this invention (repeated verification). Figure 3 Figure A shows a comparison of the concentrations of MMP1 and MMP3 proteins secreted by corneal stromal cells in keratoconus patients and healthy controls in Experiment Example 1 of this invention. Figure 3 Figure B is a comparison of the secretion levels of Caspase-1, IL-1β, and IL-18 proteins by corneal stromal cells in keratoconus patients and healthy controls in Experiment Example 1 of this invention. Figure 3 Figure C is a comparison of the secretion levels of IL-1β, TNF-α, and IL-18 proteins by corneal stromal cells in keratoconus patients and healthy controls in Experiment Example 1 of this invention; Figure 4 Figure A shows a comparison of the relative mRNA expression levels of MMP1 and MMP3 in corneal stromal cells under mechanical stress in Experiment Example 2 of this invention. Figure 4 Figure B is a comparison of the relative mRNA expression levels of NLRP3 and IL-1β in corneal stromal cells under mechanical stress in Experiment Example 2 of this invention; Figure 5 Figure A shows a comparison of the concentrations of MMP1 and MMP3 proteins secreted by corneal stromal cells under mechanical stress in Experiment Example 2 of this invention. Figure 5 Figure B is a comparison of the concentrations of Caspase-1, IL-1β, and IL-18 proteins secreted by corneal stromal cells under mechanical stress in Experiment Example 2 of this invention; Figure 6Figure A shows the Western blot results of MMP1 and MMP3 protein expression in corneal stromal cells under mechanical stress in Experiment Example 2 of this invention. Figure 6 Figure B in the figure shows the Western blot results of Piezo1-NLRP3 pathway-related proteins in corneal stromal cells under mechanical stress in Experiment Example 2 of this invention. Figure 6 C is a quantitative graph showing the relative expression levels of MMP1 and MMP3 proteins in corneal stromal cells under mechanical stress in Experiment Example 2 of this invention; Figure 6 D is a quantitative graph showing the relative expression levels of pathway-related proteins such as Piezo1 and NLRP3 in corneal stromal cells under mechanical stress in Experiment Example 2 of this invention; Figure 7 Image A shows the immunofluorescence staining of MMP1 and MMP3 in corneal stromal cells under mechanical stress in Experiment Example 2 of this invention (Control group, Stress group). Figure 7 Image B is an immunofluorescence staining image of ASC, IL-1β, NLRP3, and Piezo1 in corneal stromal cells under mechanical stress in Experiment Example 2 of this invention (Control group, Stress group). Figure 8 Figure A shows the Western blot results of Piezo1 protein expression in corneal stromal cells under siRNA treatment / untreated conditions in Example 1 of this invention. Figure 8 Figure B is a quantitative graph showing the relative expression levels of Piezo1 protein after siRNA transfection with / without siRNA treatment in Example 1 of this invention. Figure 9 Figure A shows a comparison of the relative mRNA expression levels of MMP1 and MMP3 in corneal stromal cells after Piezo1 gene silencing in Example 1 of this invention. Figure 9 Figure B is a comparison of the relative mRNA expression levels of Piezo1, ASC, and NLRP3 in corneal stromal cells after mechanical stress and Piezo1 gene silencing in Example 1 of this invention. Figure 10 Image A is a Western blot electrophoresis image of MMPs and Piezo1-NLRP3 pathway-related proteins in corneal stromal cells after mechanical stress and Piezo1 gene silencing in Example 1 of this invention. Figure 10 Figure B is a quantitative graph showing the relative expression levels of MMP1 and MMP3 proteins in corneal stromal cells after mechanical stress and Piezo1 gene silencing in Example 1 of this invention. Figure 10C is a quantitative graph showing the relative expression levels of Piezo1, NLRP3, ASC, Pro-IL-1β, and IL-1β proteins in corneal stromal cells after mechanical stress and Piezo1 gene silencing in Example 1 of this invention. Figure 11 Image A shows the immunofluorescence staining of MMP1 and MMP3 in corneal stromal cells in Example 1 of this invention (Control group, Stress group, Stress+Si-Piezo1 group). Figure 11 Image B shows the immunofluorescence staining images of ASC, IL-1β, Nlrp3, and Piezo1 in corneal stromal cells in Example 1 of this invention (Control group, Stress group, Stress+Si-Piezo1 group). Figure 12 Figure A shows a comparison of the concentrations of MMP1 and MMP3 proteins secreted by corneal stromal cells after mechanical stress and Piezo1 gene silencing in Example 1 of this invention. Figure 12 Figure B is a comparison of the concentrations of IL-1β, IL-18, and ASC proteins secreted by corneal stromal cells after mechanical stress and Piezo1 gene silencing in Example 1 of this invention. Figure 13 Figure A is a quantitative analysis of the secretion levels of MMP1 and MMP3 proteins in corneal stromal cells after treatment with the Piezo1 agonist Yoda1 and the inhibitor GsMTx4 in Example 2 of this invention, compared with the control group. Figure 13 Figure B is a quantitative analysis of the protein secretion levels of PIEZO1-NLRP3-related markers in corneal stromal cells after treatment with the Piezo1 agonist Yoda1 and the inhibitor GsMTx4 in Example 2 of this invention, compared with the control group. Figure 14 In Example 2 of this invention, image A shows the Western blot electrophoresis of MMPs and Piezo1-NLRP3 pathway-related proteins in corneal stromal cells after treatment with the Piezo1 agonist Yoda1 and the inhibitor GsMTx4. Figure 14 Figure B shows the relative expression levels of Piezo1-NLRP3 pathway-related proteins such as Piezo1 and Caspase-1 in corneal stromal cells after treatment with the Piezo1 agonist Yoda1 and the inhibitor GsMTx4 in Example 2 of this invention, compared with those in the control group. Figure 15 Image A is an immunofluorescence staining image of MMP1, MMP3 and Piezo1-NLRP3 pathway-related proteins in corneal stromal cells after treatment with the Piezo1 agonist Yoda1 and the inhibitor GsMTx4 in Example 2 of this invention, compared with the control group. Figure 15 Figure B shows the relative mRNA expression levels of MMP1 and MMP3 in corneal stromal cells after treatment with the Piezo1 agonist Yoda1 and the inhibitor GsMTx4 in Example 2 of this invention, compared with the control group. Figure 15 In Figure C, the relative mRNA expression levels of Piezo1, ASC, Nlrp3, and IL-1β in corneal stromal cells after treatment with the Piezo1 agonist Yoda1 and the inhibitor GsMTx4 in Example 2 of this invention are compared with those in the control group. Figure 16 Image A is a comparison of corneal OCT images of mouse keratoconus models (Control group, ColII group, and Treat group) in Example 3 of this invention. Figure 16 Figure B is a quantitative comparison of corneal thickness in mouse keratoconus models (Control group, ColII group, and Treat group) in Example 3 of this invention. Figure 16 The graph in C represents a comparison of the relative expression levels of MMP1 mRNA in the mouse keratoconus model (Control group, ColII group, and Treat group) in Example 3 of this invention. Figure 16 Figure D is a comparison of the relative expression levels of MMP3 mRNA in the mouse keratoconus model (Control group, ColII group, and Treat group) in Example 3 of this invention. Figure 16 E is a comparison of the relative expression levels of NLRP3 mRNA in mouse keratoconus models (Control group, ColII group, and Treat group) in Example 3 of this invention. Detailed Implementation

[0018] The following is in conjunction with the embodiments and appendices Figure 1-16 The present invention will be further described in detail below: Experimental Example Experimental Example 1 Piezo1-mediated mechanotransmission may play a central role in the development and progression of KC by promoting the aberrant release of MMPs.

[0019] First, a primary culture model of corneal stromal cells (CSCs) was established by isolating corneal tissue from keratoconus (KC) patients and healthy controls (Con) donors. Corneal tissue was treated with trypsin (Solarbio, T1300, Beijing, China) and Dispase II (Roche, 04942078001, Switzerland) to remove the epithelium, followed by digestion with type I collagenase (Collagenase I, Solarbio, C8160, Beijing, China) at 37°C for 2–4 hours to fully isolate stromal cells. After digestion, the cell suspension was filtered through a 70 μm cell sieve (Corning, USA), centrifuged to collect the pellet, and resuspended in DMEM / F12 medium (Gibco, 11320033, USA). 10% fetal bovine serum (Gibco, 10099141C) and 1% penicillin-streptomycin (HyClone, SV30010, USA) were added, and the culture was placed in a 37°C, 5% CO2 incubator (Thermo Scientific, USA). Cells were passaged to the third generation (P3) and cultured at the same density (2 × 10⁶ cells / year). 5 Cells / wells, 6-well cell plates, Corning cells were inoculated 24 hours before being used in various experiments.

[0020] To analyze gene expression, total RNA was extracted from CSCs using a Foregene RT-PCR kit (Foregene, Chengdu, China) and reverse transcribed into cDNA. Real-time quantitative PCR was performed using the SYBR Green assay system (Foregene) on an ABI StepOnePlus instrument (Applied Biosystems, USA). Results are as follows: Figure 1 As shown, the relative mRNA expression levels of MMP1, MMP3, and Piezo1 in CSCs derived from keratoconus (KC) patients were significantly higher than those in the healthy control group (Con). p <0.05), consistent with the pathological characteristics of primary cells.

[0021] Protein levels were analyzed using RIPA lysis buffer (Beyotime, P0013B, Shanghai, China) to extract total protein, and concentration was determined using the BCA method (Beyotime, P0010). 20–30 μg of protein was loaded per well. After SDS-PAGE electrophoresis, the sample was transferred to a PVDF membrane (Millipore, IPVH00010, USA) and blocked with 5% skim milk powder (Solarbio, D8340) for 1 hour. Primary antibody (anti-MMPs, Piezo1, NLRP3, IL-1β, IL-18, Abcam, USA, 1:1000) was added and incubated overnight at 4°C. After washing, HRP-labeled secondary antibody (Jackson, USA, 1:5000) was added and incubated at room temperature for 1 hour. ECL development (Beyotime, P0018AS) was performed, and grayscale analysis was performed using ImageJ software. Results are as follows: Figure 2As shown in Figures A and B, the protein levels of Piezo1, ASC, NLRP3, and IL-1β in CSCs of KC patients were also significantly elevated. p <0.05); To verify the reliability of the results, Western blot analysis was performed on corneal stromal tissue from KC patients, and the results are as follows. Figure 2 As shown in C and D, the protein expression trends of Piezo1, NLRP3, ASC, and mature IL-1β in corneal tissue are consistent with those in primary cells, further confirming the activation status of the Piezo1-NLRP3 pathway in KC. p <0.05).

[0022] For the detection of inflammatory factors, CSC culture supernatant was collected, and the levels of IL-1β and IL-18 were measured using a commercial ELISA kit (Enzyme Immunosorbent Assay Biotech, Shanghai, China). The results are as follows: Figure 3 As shown in Figure B, the KC group was significantly higher than the control group ( p <0.05).

[0023] In addition, ELISA testing was performed on tear samples from KC patients, and the results were as follows: Figure 3 As shown in Figures A and C, the levels of IL-1β, IL-18, and TNF-α were all significantly increased. p <0.05).

[0024] In summary, the results from primary cells, corneal tissue, and tear fluid samples consistently showed that KC patients exhibited MMP overexpression and Piezo1-NLRP3 signaling pathway activation, accompanied by the release of inflammatory factors. This suggests that Piezo1-mediated mechanotransmission may play a central role in the development and progression of KC by promoting abnormal MMP release, providing a molecular mechanism basis for subsequent targeted intervention of keratoconus using the Piezo1-specific inhibitor GsMTx4.

[0025] Experiment Example 2 To investigate the molecular mechanism of Piezo1 signaling dysregulation in keratoconus.

[0026] A cell traction model was established using human corneal stromal cells. After 24 hours of mechanical stretching culture, detection experiments were conducted.

[0027] Combination Figure 4 As shown in Figures A and B, qPCR analysis revealed significantly increased mRNA expression levels of matrix metalloproteinases (MMPs), NLRP3, and IL-1β.

[0028] Combination Figure 5 As shown in Figures A and B, ELISA analysis of cell culture supernatant showed that mechanical stimulation could induce increased secretion of MMPs, IL-1β, and IL-18.

[0029] Combination Figure 6 As shown, Western blotting analysis revealed that the protein expression levels of MMPs, Piezo1, NLRP3 and their downstream related markers (including ASC, caspase-1 and mature IL-1β) were significantly upregulated, and the differences were statistically significant.

[0030] Combination Figure 7 As shown, immunofluorescence analysis revealed the formation of ASC spots in corneal stromal cells after mechanical stretching, while the fluorescence intensity of Piezo1 and NLRP3 was enhanced.

[0031] Example Example 1

[0032] PIEZO1 is a key molecule in the development and progression of keratoconus. By inhibiting the PIEZO1 signaling pathway, the excessive release of MMPs induced by mechanical stimulation can be effectively suppressed.

[0033] Cell transfection Third-generation human corneal stromal cells (isolated and cultured from KC patients and healthy controls) were seeded in 6-well plates (Corning, catalog number 3516) at a density of approximately 2 × 10⁶ cells per well. 5 Cells were cultured at a density of [number] cells per cell type in DMEM / F12 medium (Gibco, catalog number 11330032) containing 10% fetal bovine serum (FBS, Gibco, catalog number 10099141C) and 1% penicillin-streptomycin (HyClone, catalog number SV30010) and incubated at 37°C in a 5% CO2 incubator (ThermoScientific). Once the cells reached 60–70% confluence, Piezo1-specific inhibitory siRNA (Yayou Biotechnology, Guangzhou, China) was added, and transfection was performed using Lipofectamine™ 3000 transfection reagent (Invitrogen, catalog number L3000001, USA) according to the manufacturer's instructions. Cells were harvested after 24–48 hours of post-transfection culture.

[0034] Western blot detection Cellular protein extraction was performed using RIPA lysis buffer (Beyotime, catalog number P0013B), and protein quantification was performed using a BCA protein assay kit (Beyotime, catalog number P0010). 30 μg of protein was taken from each well, subjected to SDS-PAGE electrophoresis, and then transferred to a PVDF membrane (Millipore, catalog number IPVH00010). The membrane was blocked with 5% skim milk powder (Solarbio, catalog number D8340) at room temperature for 1 h. Primary antibody: anti-PIEZO1 (Abcam, ab123070, 1:1000), incubated overnight at 4°C; after washing, HRP-labeled secondary antibody (Jackson, 1:5000) was added, and incubated at room temperature for 1 h. ECL development (Beyotime, catalog number P0018AS) was performed. Results are as follows. Figure 8 As shown, compared with the positive control group and the untreated group, the expression level of PIEZO1 protein in the siRNA transfection group was significantly reduced, and the difference was statistically significant, confirming that the interference was successful.

[0035] Mechanical stretching stimulation and qPCR To further validate PIEZO1 function, corneal stromal cells transfected with siRNA were placed in a Flexcell FX-5000 stretching system (Flexcell International, USA) to simulate mechanical stress (a risk factor for KC). mRNA was extracted using an RNA extraction kit (Foregene, Chengdu, China), and reverse transcription and qPCR detection were performed using a Foregene RT-PCR kit (Foregene). Results Figure 9 As shown, compared with the control group that only underwent mechanical stretching, the mRNA expression levels of MMPs, PIEZO1, NLRP3, and ASC in the PIEZO1 inhibition group were significantly downregulated. p <0.05).

[0036] Western blot protein level analysis The NLRP3 inflammasome-related protein was also detected, with antibodies sourced from Wuhan Sanying Biotechnology (China) and Abcam (UK). Results Figure 10 As shown, PIEZO1 knockdown significantly blocked mechanical stimulation-induced NLRP3 inflammasome activation and MMP protein release.

[0037] Immunofluorescence assay Cells were seeded on glass slides (NEST, catalog number 801011), transfected, and stretched. After fixation with 4% paraformaldehyde (Soleb, Beijing, China) for 15 min, permeabilized with 0.3% Triton X-100 (Sigma, catalog number T8787) for 10 min, and blocked with 5% BSA (Solarbio, SW3015) for 30 min. Primary antibodies (anti-MMPs, anti-NLRP3, anti-ASC, and anti-IL-1β, 1:200) were added, and the cells were incubated overnight at 4°C. After washing with PBS, fluorescently labeled secondary antibody (Yamei Biotechnology, Guangzhou, China, 1:500) was added, and the cells were incubated at room temperature in the dark for 1 h. Nuclei were stained with DAPI (Beyotime, C1002) for 10 min. Imaging was performed using a confocal microscope (Leica TCSSP8). Results are shown below. Figure 11 As shown, compared with the control group, the fluorescence signal intensity of MMPs, NLRP3, ASC and IL-1β was significantly reduced after PIEZO1 knockdown.

[0038] ELISA testing Cell culture supernatant was collected, and the levels of inflammatory factors were detected using a commercial ELISA kit (Enzyme Immunosorbent Assay Biotech, Shanghai, China). Results are as follows: Figure 12 As shown, the secretion of IL-1β and IL-18 was significantly reduced in the PIEZO1 inhibition group, consistent with the results of Western blotting and immunofluorescence.

[0039] The above experimental results clearly demonstrate that PIEZO1 is a key molecule in the development and progression of keratoconus. Inhibiting the PIEZO1 signaling pathway not only effectively suppresses the excessive release of MMPs induced by mechanical stimulation but also significantly reduces the associated inflammatory response. This embodiment demonstrates its crucial role by silencing Piezo1 with siRNA, while the Piezo1 inhibitor GsMTx4 can achieve similar effects by directly inhibiting Piezo1 activity and has greater clinical translational value.

[0040] Example 2

[0041] Using the drug GsMTx4 to inhibit the Piezo1 signaling pathway can reduce the release of MMPs from corneal stromal cells.

[0042] To verify the functional role of the PIEZO1 signaling pathway in the development and progression of keratoconus (KC), we treated cultured corneal stromal cells with the specific agonist Yoda1 (Selleck, catalog number S1672) and the antagonist GsMTx4 (Abcam, catalog number ab141871, final concentration 5 μM). The experiments demonstrated that GsMTx4 effectively inhibited Piezo1-mediated calcium ion influx and was non-toxic to corneal stromal cells. The specific methods are as follows: First, third-generation passaged human corneal stromal cells (isolated and cultured from KC patients and healthy controls) were seeded into 6-well plates (Corning, catalog number 3516), at a density of approximately 2 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells per cell line using DMEM / F12 medium (Gibco, catalog number 11330032) supplemented with 10% fetal bovine serum (FBS, Gibco, catalog number 10099141C) and 1% penicillin-streptomycin (HyClone, catalog number SV30010) and incubated at 37°C in a 5% CO2 incubator (ThermoScientific) for 24 hours. Subsequently, the experimental group cell culture medium was supplemented with either the PIEZO1 agonist Yoda1 (final concentration 10 μM) or the PIEZO1 antagonist GsMTx4 (final concentration 5 μM), while the control group received no treatment. All drugs were first dissolved in DMSO (Sigma, catalog number D8418) and then diluted with medium to working concentrations.

[0043] Twenty-four hours after treatment, the cell culture supernatant was collected, and the levels of inflammatory factors such as MMP1, MMP3, IL-1β, IL-18, and ASB were detected using an enzyme-linked immunosorbent assay (ELISA kit, Yamei Biotechnology). Results are as follows: Figure 13 As shown, with the activation (Yoda1 treatment) or inhibition (GsMTx4 treatment) of PIEZO1, the release of MMP3 and PIEZO1-NLRP3 related biomarkers (including NLRP3, ASC, and Caspase-1) showed a significant increasing or decreasing trend, respectively.

[0044] Meanwhile, Western blot analysis was used to detect protein levels in the treated cells. Total cell protein was extracted using RIPA lysis buffer (Beyotime Biotechnology, catalog number P0013B), and protein concentration was quantified using a BCA kit (Beyotime Biotechnology, catalog number P0010). 30 μg of protein was added to each well, followed by SDS-PAGE electrophoresis and transfer to a PVDF membrane (Millipore, catalog number IPVH00010), which was then blocked with 5% skim milk powder (Solarbio, catalog number D8340) for 1 hour. The following primary antibodies were then added: anti-PIEZO1 antibody (Abcam, ab123070, 1:1000), anti-NLRP3 antibody (Wuhan Sanying, YT3125, 1:1000), anti-MMP3 antibody (Abcam, ab52915, 1:2000), anti-ASC antibody (Abcam, ab155970, 1:1000), and anti-Caspase-1 antibody (Abcam, ab207802, 1:1000), and incubated overnight at 4°C. After washing with TBST, HRP-labeled secondary antibody (Jackson ImmunoResearch, catalog number 111-035-003, 1:5000) was added, and the membrane was incubated at room temperature for 1 hour. Development and imaging were performed using an ECL chemiluminescence kit (Beyotime, catalog number P0018AS). The results are as follows: Figure 14 As shown, when PIEZO1 is activated, the expression levels of NLRP3 inflammasome-related markers and MMPs are significantly increased; conversely, when PIEZO1 is inhibited by GsMTx4, the above indicators are significantly decreased, and the differences are statistically significant.

[0045] Immunofluorescence assays were performed to observe changes in the localization and expression of intracellular proteins. Cells were seeded on laser confocal microscopy slides (NEST, catalog number 801011) and fixed with 4% paraformaldehyde (Solarbio, P1110) for 15 minutes after drug treatment, permeabilized with 0.1% Triton X-100 (Sigma, T8787) for 10 minutes, washed with PBS, and blocked with 5% BSA (Solarbio, SW3015) for 30 minutes. Primary antibodies were then added: anti-ASC antibody (Abcam, ab155970, 1:200), anti-NLRP3 antibody (Wuhan Sanying, YT3125, 1:200), and anti-MMP3 antibody (Abcam, ab52915, 1:200), and incubated overnight at 4°C. The following day, after washing, fluorescently labeled secondary antibody (Invitrogen, Alexa Fluor 488 / 594, 1:500) was added, and the mixture was incubated at room temperature in the dark for 1 hour. Nuclei were then stained with DAPI (Beyotime, C1002) for 10 minutes. Images were acquired using a laser confocal microscope (Leica TCS SP8). Results are as follows: Figure 15As shown, when PIEZO1 is activated, ASC spots are visible in the cells, accompanied by strong positive fluorescence signals of MMPs and NLRP3; while when PIEZO1 is inhibited, the fluorescence intensity of all detection indicators is significantly reduced.

[0046] The above results indicate that activation of PIEZO1 signaling can induce the activation of MMPs and the NLRP3 inflammasome, thereby promoting the release of inflammatory factors. Inhibition by the PIEZO1 antagonist GsMTx4 significantly blocks this process and reduces the level of inflammatory response, consistent with the results of qPCR experiments. Activation of Piezo1 promotes the development of keratoconus. GsMTx4, by inhibiting Piezo1 activity, significantly reduces MMP1 / MMP3 secretion and NLRP3 inflammasome activation, and its inhibitory effect is superior to the short-term intervention of Piezo1 gene silencing in Example 3.

[0047] Example 3

[0048] The effects of the Piezo1 inhibitor GsMTx4 have been confirmed in animal models.

[0049] The mice were divided into three groups: Control group (normal mice), ColII group (type II collagenase treatment group), and Treat group (ColII modeling group treated with 5 μM GsMTx4 eye drops).

[0050] Healthy C57BL / 6 mice (approximately 18–22 g) were housed in a clean-grade animal facility under normal temperature and humidity conditions, with free access to food and water. In the ColII group, mice were fixed on an operating table under isoflurane anesthesia, and the corneal epithelium in the central region of the right cornea was gently removed using a sterile corneal curette. Subsequently, 10 μL of type II collagenase solution (final concentration 2 μM) was instilled onto the corneal surface and left for approximately 30 minutes to disrupt the corneal stromal structure and induce keratoconus-like changes. After treatment, residual enzyme solution was removed by rinsing with sterile PBS (Biosharp, catalog number BL601A). In the Treat group, under the same modeling conditions, mice were additionally given eye drops containing the mechanosensitive ion channel inhibitor GsMTx4 (final concentration 5 μM, diluted with sterile PBS), one drop (approximately 10 μL) five times daily. The control group received only an equal volume of PBS solution. Corneal thickness was measured before modeling and at 0, 3, 7, 14, and 28 days after modeling. Mice were anesthetized and placed in a small animal OCT imaging system to measure central corneal thickness. Scans were performed using a Leica Envisu R2200SD-OCT (Leica Microsystems, Germany), and images were saved for subsequent analysis. Results are as follows: Figure 16As shown, the corneal thickness of mice in the control group with keratoconus gradually thinned over time, while the corneal thickness of mice in the GsMTx4 treatment group did not show a significant thinning trend throughout the observation period. In molecular-level detection, total RNA was extracted from mouse corneal tissue using TRIzol reagent, and cDNA was synthesized using a reverse transcription kit from TransGenBiotech (Wuhan, China). Amplification and detection were then performed using qPCRSuperMix reagent provided by TransGenBiotech on a real-time quantitative PCR instrument. The results showed that the mRNA expression level of MMPs in the corneal stroma of the treatment group was significantly lower than that of the control group, and the expression level of the inflammatory factor NLRP3 was also significantly reduced. These results indicate that GsMTx4 can effectively reduce the release of MMPs in the corneal stroma of keratoconus model mice and alleviate the inflammatory response, thereby delaying the dissolution and thinning of the corneal stroma, demonstrating a clear therapeutic effect on keratoconus.

[0051] Combination Figure 16 In mice in the C and Col II groups, MMP1 mRNA expression was significantly upregulated compared to the Control group due to the destruction of corneal stroma structure by collagenase. In contrast, the relative expression level of MMP1 mRNA in the cornea of ​​mice treated with GsMTx4 was significantly restored to near normal levels, confirming that GsMTx4 can effectively inhibit the transcriptional activity of the MMP1 gene in the corneal stroma.

[0052] Combination Figure 16 In the middle D and Col II groups, MMP3 expression was activated due to the pathological progression of keratoconus, and its relative expression level was significantly upregulated compared with the Control group. After intervention with GsMTx4, the relative expression level of MMP3 mRNA in the Treat group was significantly reduced and returned to normal level, further verifying that the inhibitory effect of GsMTx4 on matrix metalloproteinase family (MMPs) is broad-spectrum, rather than targeting only MMP1.

[0053] Combination Figure 16 In the E and Col II groups, chronic inflammation was caused by corneal stromal damage, and NLRP3 mRNA expression was significantly higher than that in the Control group. After treatment with GsMTx4, the relative expression of NLRP3 mRNA in the Treat group was significantly reduced and returned to normal levels. This indicates that GsMTx4 can not only inhibit the expression of MMPs, but also block the inflammasome activation pathway by downregulating NLRP3 gene transcription, thereby reducing the inflammatory response of corneal tissue from the source.

[0054] The above are all preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Therefore, all equivalent changes made in accordance with the structure, shape and principle of the present invention should be covered within the scope of protection of the present invention.

Claims

1. The use of a Piezo1 inhibitor in the preparation of a drug for the treatment of keratoconus.

2. The use of a piezo1 inhibitor according to claim 1 in the preparation of a drug for treating keratoconus, characterized in that, The piezo1 inhibitor is GsMTx4.

3. A drug for treating keratoconus, characterized in that, The drug contains a pharmacologically effective concentration of GsMTx4.

4. The medicament for treating keratoconus according to claim 2, characterized in that, The drug can inhibit the release of matrix metalloproteinases in corneal stromal cells and the activation of the NLRP3 inflammasome, thereby reducing the secretion of inflammatory factors.

5. The medicament for treating keratoconus according to claim 2, characterized in that, The medication in question is an eye drop.

6. The medicament for treating keratoconus according to claim 4, characterized in that, The concentration of GsMTx4 in the eye drops is 1-10 μM.

7. The medicament for treating keratoconus according to claim 5, characterized in that, The concentration of GsMTx4 in the eye drops is 5 μM.

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