Application of costunolide in the preparation of drugs for preventing posterior capsule opacification

Abstract

This invention provides the application of costunolide in the preparation of a drug for preventing posterior capsule opacification. Costunolide effectively reduces posterior capsule opacification after extracapsular lens extraction (ECLE) and exhibits significant anti-inflammatory and anti-fibrotic effects in both in vitro and in vivo models. Mechanistic studies have identified matrix metalloproteinase 8 (MMP8) as a key downstream effector of costunolide, confirming that costunolide inhibits MMP8 expression, thereby blocking the transformation of lens epithelial cells (LECs) into mesenchymal cells and subsequent fibrotic processes. This lays a solid foundation for developing targeted therapies for posterior cataract, a common surgical complication.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical application technology, and to the use of costunolide in the preparation of medicaments for preventing posterior capsule opacification, particularly the use of costunolide in the preparation of medicaments for preventing, treating, improving or preventing posterior capsule opacification or fibrosis or inflammation associated with eye-related diseases or eye-related surgeries. Background Technology

[0002] Cataracts are the leading cause of blindness worldwide, and their incidence is rising with the aging population. Notably, this disease still affects a significant proportion of the working-age population, severely impacting individual productivity and imposing a heavy economic burden on society. Surgery is currently the most effective treatment for cataracts; however, postoperative complications remain a significant concern. Posterior capsular opacity (PCO), the most common postoperative complication, has become a major cause of postoperative visual impairment. Clinical data show that the incidence of PCO can reach 6% six months post-surgery, rising to 50% nine years later, and reaching as high as 100% in children. The mechanism of PCO stems from the inflammatory response of residual lens epithelial cells (LECs) within the capsular bag and the wound healing process. These activated LECs undergo a series of pathological changes, including abnormal proliferation, migration, epithelial-mesenchymal transition (EMT), collagen deposition, and fibrotic regeneration. Throughout the wound healing process, cellular behavior undergoes significant changes, characterized by a marked upregulation of key EMT markers—including α-smooth muscle actin (α-SMA), fibronectin (FN), and type I collagen (Col I)—concurrently with a downregulation of characteristic epithelial proteins. Transforming growth factor-β2 (TGF-β2), an intrinsic component of the aqueous humor and a major isoform of the anterior segment, plays both physiological and pathological roles in the eye. Under physiological conditions, it helps maintain the eye's immune immunity; however, its significant upregulation after cataract surgery triggers pathological changes. This surge activates the TGF-β / Smad signaling pathway, which becomes a core driver of EMT and subsequent PCO development. Extracapsular lens extraction (ECLE) models effectively simulate the EMT process induced by inflammation and surgical trauma. In this model, a series of sequential responses were observed after the removal of lens fibers: the levels of inflammatory cytokines peaked within 24 hours, followed by activation of the TGF-β / p-Smad3 signaling pathway at 48 hours, ultimately leading to the maximum increase in the expression of α-SMA and FN on day 5.

[0003] Micheliolide (CAS 68370-47-8), also known as Micheliolide, has the following structural formula: This compound is mainly derived from the root bark of plants such as Michelia champaca (Magnolia family), and can also be isolated from Tanacetum parthenium (Asteraceae family). It belongs to the sesquiterpene lactone class of compounds. Costus lactones possess a wide range of biological activities, primarily exerting their pharmacological effects by regulating multiple signaling pathways, including antitumor, anti-inflammatory, immunomodulatory, neuroprotective, and organ-protective effects.

[0004] Currently, there is no known application of costunolide in the preparation of drugs for the prevention and / or treatment of secondary cataracts, especially for the prevention and / or treatment of posterior capsule opacification. Therefore, developing the application of costunolide in the preparation of drugs for the prevention and / or treatment of posterior capsule opacification is of great significance. Summary of the Invention

[0005] Based on this, this invention proposes the application of costunolide in the preparation of drugs for preventing posterior capsule opacification. Costunolide effectively reduces posterior capsule opacification after ECLE surgery, exhibiting significant anti-inflammatory and anti-fibrotic effects in both in vitro and in vivo models. Mechanistic studies have identified matrix metalloproteinase 8 (MMP8) as a key downstream effector of costunolide, confirming that costunolide inhibits MMP8 expression, thereby blocking the transformation of LECs into mesenchymal cells and subsequent fibrosis, laying a solid foundation for developing targeted therapies for this common surgical complication.

[0006] In a first aspect, the present invention provides the use of costunolide in the preparation of a medicament for the prevention, treatment, improvement or prevention of posterior capsule opacification or fibrosis or inflammation associated with eye-related conditions or eye-related surgeries.

[0007] In some embodiments, the fibrosis or inflammation associated with eye-related conditions or eye-related surgery is selected from cataracts, secondary cataracts after extracapsular lens extraction, secondary cataracts, posterior capsule fibrosis, congenital cataracts, cortical opacities, or posterior subcapsular cataracts.

[0008] In some embodiments, the effective concentration of the drug is 1.25-10 mM, preferably 1.25 mM, 2.5 mM, 5 mM and 10 mM.

[0009] In some embodiments, the drug further includes a matrix metalloproteinase-8 inhibitor (MMP8 inhibitor), such as M8I (a specific inhibitor of MMP-8, MCE (catalog number HY-N 0578R)).

[0010] In some embodiments, the drug blocks the transformation of lens epithelial cells (LECs) into mesenchymal cells and the subsequent fibrotic process by inhibiting the expression of matrix metalloproteinase 8.

[0011] In some embodiments, the drug can effectively inhibit the epithelial-mesenchymal transition (EMT) of lens epithelial cells and the epithelial-mesenchymal transition process of the capsular bag after ECLE surgery.

[0012] In some embodiments, the drug can inhibit the proliferation and migration of anterior capsule epithelial cells after ECLE surgery.

[0013] In some embodiments, the drug can effectively inhibit the expression of EMT protein markers α-smooth muscle actin (α-SMA), fibronectin (FN), and type I collagen (Col I) in LECs.

[0014] In some embodiments, the drug can suppress the inflammatory response after extracapsular lens extraction.

[0015] In some embodiments, the drug achieves inflammation suppression by inhibiting the expression of inflammatory markers CXCL1, COX-2, G-CSF, and S100A9.

[0016] In some embodiments, the drug can alleviate the migration-promoting phenotype of LECs induced by transforming growth factor-β2 (TGF-β2).

[0017] In some embodiments, the drug effectively inhibits the expression and level of EMT marker proteins (α-SMA, Col I, FN).

[0018] In some embodiments, the drug inhibits the EMT process of LCEs by downregulating MMP8.

[0019] In some embodiments, the drug is administered by injection into the eye, vitreous body, intraocularly, in the anterior chamber, within a lesion, subconjunctivally, or subfascial, or by drug delivery via eye drops, sprays, adhesives, or implants, or by intratubal delivery.

[0020] In some embodiments, the drug is administered via intra-atrial injection immediately after ECLE surgery.

[0021] Beneficial effects

[0022] (1) Study on the protective effect and mechanism of costunolide on PCO. This invention systematically evaluated the protective effect and molecular mechanism of costunolide on secondary cataract through in vitro and in vivo experiments. In vivo experiments used an extracapsular lens extraction (ECLE) model, with the drug administered via intra-anterior chamber injection; in vitro experiments used a cell model of TGF-β2-induced epithelial-mesenchymal transition in lens epithelial cells. The cytotoxicity of the drug was assessed by TUNEL staining and Alizarin Red staining, and the expression changes of EMT and inflammatory markers were detected by Western blot, qRT-PCR, and immunofluorescence. The proliferation and migration ability of LECs were assessed by HE staining, Transwell migration assay, and wound healing assay. The experimental results showed that costunolide significantly inhibited the inflammatory response, EMT marker expression, and proliferation and migration ability of LECs after ECLE in both animal models and cell experiments. To further elucidate the mechanism, RNA sequencing analysis was performed on lens capsule tissue at 0 hours, 24 hours, and 5 days after ECLE. Transcriptome analysis revealed that matrix metalloproteinase 8 (MMP8) expression was significantly upregulated 24 hours post-surgery, and costunolide administration effectively inhibited this induction. In vitro experiments further confirmed that the combination of costunolide and the MMP8 inhibitor (M8I) synergistically inhibited the expression of EMT-related proteins in LECs.

[0023] (2) Application of costunolide in preventing posterior capsule fibrosis. This invention reveals the mechanism by which costunolide targets and inhibits MMP8 expression, blocking the transformation of LECs into mesenchymal cells and their subsequent fibrotic process, providing a new treatment strategy for preventing or delaying posterior capsule fibrosis. This discovery lays an important foundation for developing targeted drugs for posterior cataract, a common surgical complication, and has significant clinical application value. Attached Figure Description

[0024] Figure 1 Figure 1 shows the Western blot and quantitative results of EMT markers in the pouch of mice after ECLE surgery at different time points. (A) Western blot; (B) Quantitative results of E-cadherin; (C) Quantitative results of fibronectin; (D) Quantitative results of type I collagen; (E) Quantitative results of α-smooth muscle actin.

[0025] Figure 2 Immunofluorescence staining of α-SMA (green) in the cyst bag of mice on day 5 after ECLE combined with anterior chamber injection of gradient concentrations of costunolide, scale bar = 20 μm.

[0026] Figure 3 Alizarin red staining images of corneal endothelial cells at 0 hours and 5 days after ECLE combined with anterior chamber injection of normal saline or 5 mM costunolide, scale bar = 50 μm.

[0027] Figure 4 TUNEL staining (green) of the retina on day 5 after ECLE combined with anterior chamber injection of saline or 5 mM costunolide, scale bar = 50 μm.

[0028] Figure 5 This image shows the expression of E-cadherin, α-SMA, FN, and Col I in the capsule using time-series immunofluorescence analysis. Images were acquired at 0, 24, 48 hours, and day 5. Scale bar = 20 μm. (A) Control group; (B) Experimental group.

[0029] Figure 6 The diagram shows the inhibitory effects of costunolide on postoperative EMT and cellular responses. (A) The protein expression of EMT markers in the pouches of ECLE mice treated with saline or costunolide was assessed by Western blotting. (B) The expression results of fibronectin were shown. (C) The expression results of α-smooth muscle actin were shown. (D) The transcriptional level of type I collagen was assessed by qRT-PCR. (E) The transcriptional level of fibronectin was assessed by qRT-PCR. (F) The transcriptional level of α-smooth muscle actin was assessed by qRT-PCR.

[0030] Figure 7 HE staining histological analysis shows cell density and migration status in the cysts under various treatment conditions. Scale bar = 0.2 / 0.5 mm.

[0031] Figure 8 Immunofluorescence analysis of key inflammatory factors (CXCL1, COX-2, G-CSF, S100A9) in the cyst bag 24 hours after ECLE surgery and after treatment with costunolide / saline. Scale bar = 20 μm.

[0032] Figure 9 The diagrams show the inhibitory effects of costunolide on the transcriptional levels of these inflammatory mediators, as confirmed by qRT-PCR. (A) Inhibition of cyclooxygenase 2; (B) Inhibition of chemokine ligand 1; (C) Inhibition of granulocyte colony-stimulating factor; (D) Inhibition of S100 calcium-binding protein A9.

[0033] Figure 10The inhibitory effect of costunolide on TGF-β2-driven LEC responses. (A) Dose-dependent effect of costunolide on LEC activity. (B)-(C) mRNA (B) and protein (C) expression levels of key EMT-related genes in LECs pretreated with or without costunolide under TGF-β2 stimulation; (B1) qRT-PCR image of fibronectin, (B2) qRT-PCR image of type I collagen, (B3) qRT-PCR image of α-smooth muscle actin; (C1) Western blot results, (C2) results for fibronectin, (C3) results for type I collagen, (C4) results for α-smooth muscle actin. (D) Transwell assay to assess vertical migration, scale bar = 100 μm. (E) Scratch assay to monitor horizontal migration, scale bar = 500 μm. (F) Quantitative analysis of the number of migrating cells. (G) Quantitative analysis of the scratch healing rate.

[0034] Figure 11 This image shows the attenuation of EMT marker expression in LECs induced by costunolide under TGF-β2 treatment. In (A), a representative immunofluorescence image shows the expression of type I collagen, FN, and α-SMA (green) in TGF-β2-treated LECs with or without costunolide. DAPI (blue) was used for nuclear staining. Scale bar = 100 μm. (B), (C), and (D) represent the quantitative analysis of immunofluorescence intensity of each marker at 0, 24, and 5 days, respectively.

[0035] Figure 12 PCA analysis of FPKM values ​​in each group of samples during transcriptome analysis of genes related to costunolide regulation after ECLE surgery in mice.

[0036] Figure 13 This is a volcano plot of differentially expressed genes (DEGs) between the NS-24h and NS-0h groups during transcriptome analysis of genes related to costunolide regulation after ECLE surgery in mice. DEGs significantly upregulated in the NS-24h group (padj ≤ 0.05 and |log2(fold change)| ≥ 1) are marked in red, and significantly downregulated DEGs are marked in blue. Genes with no significant difference are marked in gray.

[0037] Figure 14 In the transcriptome analysis of genes related to costunolide regulation after ECLE surgery in mice, KEGG enrichment analysis of DEGs in the NS-24h and NS-0h groups was performed, and the top 20 pathways were displayed in order of Padj value.

[0038] Figure 15A figure showing the transcriptome analysis of genes related to costunolide regulation after ECLE surgery in mice. (A) Heatmap of EMT and inflammation-related genes in the NS-5d, NS-24h, and NS-0h groups. (B) Volcano plot of DEGs in the NS-5d and NS-0h groups. Genes significantly upregulated in the NS-24h group (padj ≤ 0.05 and |log2(fold change)| ≥ 1) are highlighted in red, and genes significantly downregulated are shown in blue. Genes with no significant difference are shown in gray.

[0039] Figure 16 In the transcriptome analysis of genes related to costunolide regulation after ECLE surgery in mice, KEGG enrichment analysis of DEGs in the NS-5d and NS-0h groups was performed, and the top 20 pathways were displayed in order of Padj value.

[0040] Figure 17 Two figures show the transcriptomic analysis of genes related to costunolide regulation after ECLE surgery in mice. (A) Venn diagram of the number of overlapping genes between the NS-24h group and the NS-0h group and the MCL-24h group. (B) Heatmap of 23 DEGs, showing that these genes are upregulated in the NS group and downregulated in the MCL treatment group.

[0041] Figure 18 This is a DEG volcano plot comparing the MCL-24h group and the NS-24h group during transcriptome analysis of genes related to costunolide regulation after ECLE surgery in mice. Red represents upregulated genes, blue represents downregulated genes, and gray represents genes with no significant difference.

[0042] Figure 19The following images illustrate the effects of costunolide downregulating MMP8 in EMT. (A) MMP8 mRNA expression in bag tissue injected with saline or costunolide 24 hours after ECLE surgery. (B) Western blot image of MMP8 in bag tissue injected with saline or costunolide 24 hours after ECLE surgery. (C) Protein expression of MMP8 in bag tissue injected with saline or costunolide 24 hours after ECLE surgery. (D) MMP8 expression in LECs pretreated with or without costunolide (5 μmol / L) and then treated with TGF-β2 (10 ng / mL, 48 h), as shown by qRT-PCR. (E) MMP8 expression in LECs pretreated with or without costunolide (5 μmol / L) and then treated with TGF-β2 (10 ng / mL, 48 h), as shown by Western blot. (F) Expression of MMP8 in LECs with or without pretreatment with costunolide (5 μmol / L) followed by treatment with TGF-β2 (10 ng / mL, 48 h), as shown by protein level expression graph. (G) Activity of LECs treated with increasing concentrations of M8I. (H)-(K) Protein levels of EMT markers in LECs treated with costunolide and M8I under TGF-β2 stimulation. Representative blot maps and quantitative analysis results are presented.

[0043] Figure 20 Co-localization of α-SMA and MMP8 in LECs. (A) Co-immunofluorescence staining of α-SMA (red) and MMP8 (green) in TGF-β2-induced LECs treated with costunolide alone or in combination with M8I, scale bar = 100 μm. (B) Quantitative analysis of immunofluorescence intensity of MMP8. (C) Quantitative analysis of immunofluorescence intensity of α-SMA.

[0044] Figure 21 This diagram illustrates the mechanism by which MCL alleviates post-cataract surgery inflammation and inhibits lens epithelial cell proliferation, migration, and EMT by targeting MMP8. In the diagram: PCS: Post-cataract surgery.

[0045] Terminology Explanation

[0046] Certain embodiments of the invention will now be described in detail, examples of which are illustrated by the accompanying structural and chemical formulas. The invention is intended to cover all alternatives, modifications, and equivalents, all of which are included within the scope of the invention as defined in the claims. Those skilled in the art will recognize that many similar or equivalent methods and materials can be used to practice the invention. The invention is by no means limited to the methods and materials described herein. In the event that one or more of the incorporated documents, patents, and similar materials differ from or contradict this application (including, but not limited to, defined terminology, application of terminology, described techniques, etc.), this application shall prevail.

[0047] It should be further appreciated that certain features of the invention, for clarity, have been described in multiple independent embodiments, but may also be provided in combination in a single embodiment. Conversely, various features of the invention, for brevity, have been described in a single embodiment, but may also be provided individually or in any suitable sub-combination.

[0048] Unless otherwise stated, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. All patents and publications related to this invention are incorporated herein by reference in their entirety.

[0049] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0050] In the following content, all numbers disclosed herein, whether or not they use words such as "approximately" or "about," are approximate values. The value of each number may vary by 1%, 2%, 5%, 7%, 8%, 10%, 15%, or 20%. Whenever a number with a value of N is disclosed, any numbers with values ​​of N+ / -1%, N+ / -2%, N+ / -3%, N+ / -5%, N+ / -7%, N+ / -8%, N+ / -10%, N+ / -15%, or N+ / -20% will be explicitly disclosed, where "+ / -" indicates addition or subtraction. Detailed Implementation

[0051] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0052] All reagents used in this invention can be purchased commercially or prepared by the methods described in this invention.

[0053] The products, companies, and catalog numbers are as follows: SRA01 / 04 cell line, Cyconruc Biosciences (Nanjing, China); Fetal Bovine Serum (FBS), Ecosai (Suzhou, China), Cat. FSP500; Penicillin-Streptomycin (Bispecific Antibody), Beyotime (Shanghai, China), Cat. C0222; Micheliolide, MedChemExpress, Cat. HY-N0847; TGF-β2, Proteintech (Chicago, USA), Cat. HZ-1092; MMP8 inhibitor, MedChemExpress, Cat. HY-N0578R; CCK-8 reagent, Ekoto (Guangzhou, China), Cat. EK-5103; Trizol, Thermo Fisher Scientific (Massachusetts, USA), Cat. 15596018; cDNA synthesis premix kit, Yisheng Biotechnology (Shanghai, China), Cat. 11141; SYBR Green premix, Yisheng Bio (Shanghai, China), Cat. 11202; RIPA lysis buffer, Numer Bio (Suzhou, China), Cat. WB3100; Protease / phosphatase inhibitor mixture, Numer Bio (Suzhou, China), Cat. P002; BCA protein quantification kit, Thermo Fisher Scientific (Massachusetts, USA), Cat. 23227; ECL chemiluminescence assay reagent, Numer Bio (Suzhou, China), Cat. P10100; Ocular fixative, Xavier Biotech (Wuhan, China), Cat. G1109; Paraformaldehyde, Xavier Biotech (Wuhan, China), Cat. G1101; DAPI (nuclear dye), Solarbio (Beijing, China), Cat. C0065; Anti-fluorescence quenching mounting medium, Ecoto (Guangzhou, China), Cat. ES-8312; Confocal microscope, Carl Zeiss (Göttingen, Germany); Transwell chamber, Corning (New York, USA), Cat. CLS3422; Crystal violet, Ecoto (Guangzhou, China), Cat. ES-8107; Alizarin Red, Solarbio (Beijing, China), Cat. G1452; TUNEL reagent, Yulanda (Suzhou, China), Cat. T6013.

[0054] Abbreviations

[0055] PCO: Posterior capsule opacification; Col I: Type I collagen; Col IV: Type IV collagen; DEGs: Differentially expressed genes; ECLE: Extracapsular lens extraction; ECM: Extracellular matrix; EMT: Epithelial-mesenchymal transition; FBS: Fetal bovine serum; Fn: Fibronectin; FPKM: Number of fragments per kilobase transcript per million mapping reads; GBM: Glioblastoma multiforme; IL-1β: Interleukin-1β; IL-6: Interleukin-6; M8I: MMP8 inhibitor; MCP-1: Monocyte chemoattractant protein-1; MMP-9: Matrix metalloproteinase-9; MMP8: Matrix metalloproteinase-8; OCT: OCT embedding agent (optimal cleavage temperature compound); PCA: Principal component analysis; qRT-PCR: Real-time quantitative polymerase chain reaction; TGF-β2: Transforming growth factor-β2; TNC: Tenosynovin; TNF-α: Tumor necrosis factor-α; WB: Western blotting; α-SMA: α-smooth muscle actin.

[0056] Example 1

[0057] 1 Extracapsular lens extraction model

[0058] Eight-week-old mice were deeply anesthetized with 1% sodium pentobarbital and mydriatic with tropicamide. After 3-5 minutes, the corneal reflex was confirmed to be absent. At the start of the surgery, a central incision was made in the cornea using a 15° ophthalmic scalpel; this incision also served to open the anterior lens capsule. The lens contents were then dissected and removed via saline irrigation. After ensuring the removal of any remaining cortex, the corneal incision was sutured with 10-0 sutures. At the end of the surgery, approximately 5 μl of saline or costunolide was injected into the anterior chamber using a 31-G insulin needle. Tobramycin-dexamethasone eye ointment was applied to the conjunctival sac, the ears were tagged, and the mice were housed in an SPF-grade, temperature- and humidity-controlled isolation environment. The eyeballs and lens capsules were removed at 0, 24, 48, and 5 days post-ECLE for subsequent experiments. All animal procedures were performed in accordance with the guidelines of the American Association for Vision and Ophthalmology Research and were approved by the Ethics Review Committee of Xiangya Hospital, Central South University.

[0059] 2. Cell Culture and Processing

[0060] SRA01 / 04 cells were cultured in a humidified incubator at 37°C and 5% CO2 using medium supplemented with 20% fetal bovine serum (FBS) and 100 U / mL penicillin-streptomycin. To analyze the effect of costunolide on TGF-β2-induced epithelial-mesenchymal transition (EMT) in vitro, cells with 50-60% confluence were first exposed to a 5 μmol / L costunolide solution, followed by overnight serum-free starvation. Subsequently, before cell collection for experiments, they were treated in serum-free medium containing 10 ng / mL TGF-β2 for 48 hours. To investigate the role of MMP8 in the effect of costunolide on the in vitro EMT model, cells were pretreated with a 100 μmol / L MMP8 inhibitor (M8I) in combination with costunolide, starved overnight when cell confluence reached 50-60%, and then treated in serum-free medium containing 10 ng / mL TGF-β2 for 48 hours before collection for experiments.

[0061] 3. Cell viability assay

[0062] To evaluate the cytotoxicity of costunolide or M8I (MMP8 inhibitor) solutions, 5 × 10 3 Cells were seeded in 96-well plates and cultured for 12 hours. Subsequently, the cells were treated with serum-free medium containing serially diluted MCL or M8I for 48 hours. After incubation, the original medium was discarded, and 10% CCK-8 solution was added. The cells were then incubated in the dark for 2–4 hours, followed by absorption measurement at 450 nm.

[0063] 4. Real-time quantitative polymerase chain reaction (qRT-PCR)

[0064] Total RNA was extracted using the Trizol method. The concentration and purity of RNA in the extracted samples were quantitatively analyzed using a spectrophotometer, and the optimal sample was selected for reverse transcription. cDNA was synthesized using a cDNA synthesis premix kit with 1 μg of RNA as a template. Subsequently, the target gene was amplified using SYBR Green premix. The primer sequences used for mRNA detection are listed in Table 1.

[0065] Table 1 Primer sequences used in real-time quantitative PCR

[0066]

[0067] 5. Western blot analysis (WB)

[0068] Cell and lens capsule samples were collected at specific time points. After collection, samples were lysed using RIPA lysis buffer containing 1% protease and phosphatase inhibitors to extract total protein, and protein concentration was determined by the BCA method. Protein samples were separated by SDS-PAGE (8% or 10% gel), transferred to PVDF membranes, and blocked with 5% skim milk or BSA. The membranes were incubated overnight at 4°C with primary antibodies (shown in Table 2), washed with PBST, and then incubated with HRP-labeled secondary antibodies. Protein bands were visualized using ECL chemiluminescent substrates, and signals were captured. Quantitative analysis was performed using ImageJ software to measure band intensity (grayscale value).

[0069] Table 2. Primary antibody dilution ratios for Western blotting (WB) and immunofluorescence (IF).

[0070]

[0071] 6. Hematoxylin-eosin staining

[0072] After anesthetizing the mice, the eyelids were cut open and removed, the muscles and connective tissues were separated and incised, and the optic nerve was severed while maintaining the integrity of the eyeball. The eyeball was then immersed in an eye fixation solution for approximately 24 hours. The fixed eyeball was removed and dehydrated using a gradient of ethanol, followed by treatment twice with pure xylene until the tissue became translucent. The tissue was then immersed in pure paraffin 2-3 times (30-60 minutes each time) for paraffin embedding, and embedded using an embedding machine, and sectioned into 4 μm thick serial sections. The serial sections were flattened in 40°C warm water, lifted with a detachable slide, and baked at 60°C for 2-4 hours. The dried sections were first dewaxed with xylene, then hydrated using a gradient of decreasing ethanol concentrations. Subsequently, they were stained with hematoxylin for 5 minutes, dehydrated by reverse ethanol treatment, and counterstained with eosin for the same time. Finally, the sections were cleared with xylene, mounted with neutral resin, and observed under an optical microscope.

[0073] 7. Immunofluorescence staining

[0074] After euthanizing mice, the excised eyeballs were embedded in OCT embedding medium, flash-frozen on dry ice, and stored at -80°C. Serial frozen sections of 16 μm thickness were prepared, air-dried, and washed with PBS to remove residual OCT. Sections were fixed with 4% paraformaldehyde for 15 minutes, washed three times with PBS (5 minutes each time), permeabilized with 0.3% PBST for 15 minutes, and then blocked with 5% BSA at room temperature for 30 minutes. Sections were incubated with primary antibody overnight at 4°C. After washing three times with PBS (10 minutes each time), sections were incubated with fluorescently labeled secondary antibody (1:1000 dilution) at room temperature in the dark for 2 hours. Finally, cell nuclei were counterstained with DAPI, and slides were mounted with anti-fluorescence quenching mounting medium and observed under a confocal microscope.

[0075] 8. Cell Scratch Assay

[0076] After complete cell confluence, a sterile 10 μL pipette tip was used to make a scratch along the longitudinal axis of the culture plate, followed by washing three times with PBS to remove detached cells. Each group was then treated accordingly and cultured further. Images were taken at 0, 24, and 48 hours post-scratching to assess cell migration ability. The scratch healing rate was calculated using ImageJ software using the following formula: Scratch healing rate = (0-hour scratch area - [X]-hour scratch area) / 0-hour scratch area × 100%.

[0077] 9 Transwell transfer experiments

[0078] After treatment, cells were collected and resuspended in serum-free medium. The density was 2 × 10⁶ cells / day. 4 Cell suspension was added to the upper chamber of the Transwell. 600 μL of culture medium containing 20% ​​serum was added to the lower chamber. After culturing for 48 hours, the migrated cells were fixed with 4% paraformaldehyde (20 minutes). Unmigrated cells on the upper side of the membrane were wiped away with a cotton swab, and the migrated cells on the lower side of the membrane were stained with 0.1% crystal violet (30 minutes). After rinsing with PBS, images of the stained cells were taken under a microscope.

[0079] 10 Alizarin Red Staining

[0080] Five days after establishing the ECLE (extracapsular lens extraction) model, the cornea was carefully and completely removed by incision along the limbus. The tissue was laid flat on a glass slide with the endothelial side facing up and stained with 1% Alizarin Red for 1 minute. It was then gently rinsed two to three times with physiological saline until no residual dye remained. The morphology and arrangement of the corneal endothelial cells were observed under a microscope.

[0081] 11 TUNEL staining experiment

[0082] According to the kit instructions, the dewaxed sections were first treated with 20 μg / mL proteinase K at room temperature for 20 minutes to increase permeability. After equilibration in buffer for 5 minutes, TUNEL reaction mixture (TdT enzyme and buffer at a ratio of 1:49) was added, and the sections were incubated in a dark, humid incubator for 2 hours. The reaction was terminated by washing three times with PBS for 5 minutes each time, and finally counterstained with DAPI for 5 minutes.

[0083] 12 RNA Sequencing and Data Analysis

[0084] Lens capsule tissue was collected from mice at 0 hours, 24 hours, and 5 days post-ECLE surgery. Two capsule tissue samples were combined as a biological replicate for RNA extraction. NanoPhotometer was used. ®RNA concentration and purity were determined, and RNA integrity was assessed using an Agilent 2100 Bioanalyzer (RIN>7 required). Library construction, library detection, sequencing, and data quality control were all performed according to the methodologies and procedures outlined in previous studies (Fan et al., 2024a).

[0085] 13 Statistical Analysis

[0086] Statistical analysis was performed using GraphPad Prism 10 software. Data from at least three independent experiments are presented as mean ± standard deviation. One-way ANOVA was used for comparisons among multiple groups. A p-value less than 0.05 was considered statistically significant, and the significance levels were indicated as follows: .

[0087] result

[0088] 1. Costunolide inhibits the proliferation, migration, and epithelial-mesenchymal transition of lens epithelial cells in vivo.

[0089] This study evaluated the expression of EMT-related proteins at different time points after ECLE surgery. Figure 1 This study confirmed that the epithelial-mesenchymal transition (EMT) process of lens epithelial cells (LECs) within the capsular bag develops over time. Based on the applicant's previous research (Fan et al., 2024b; Jiang et al., 2018), it has been determined that the EMT process peaks on the fifth postoperative day. Therefore, in the subsequent comparative analysis of experimental groups, the fifth postoperative day will be selected as the observation endpoint.

[0090] Following ECLE surgery, MCL at doses of 1.25 mM, 2.5 mM, 5 mM, and 10 mM was injected into the anterior chamber to determine the optimal concentration for inhibiting EMT in LECs. Immunofluorescence staining on day 5 showed that α-SMA expression in the cyst pockets of different concentration groups exhibited a concentration-dependent modulation. 5 mM was determined to be the minimum effective dose because it resulted in the weakest α-SMA positive staining. Figure 2 Therefore, it was selected as the concentration for subsequent research.

[0091] Simultaneously, retinal cell apoptosis was detected using the TUNEL assay. Figure 4 The results showed that, 5 days after anterior chamber injection of 5 mM costunolide, the number of apoptotic positive cells in the retina did not increase compared to the control group. Alizarin red staining was used to observe morphological changes in corneal endothelial cells. Figure 3 The results showed that the corneal endothelial cells were neatly arranged with clear boundaries. Notably, the toxicity of 5 mM costunolide was negligible, and therefore it was selected for subsequent in vivo experiments.

[0092] The expression levels of E-cadherin (an epithelial marker, E-cadherin) and α-SMA, FN, and Col I (an EMT marker) in the cyst were monitored by time-series immunofluorescence analysis (0h, 24h, 48h, 5d). Figure 5 Following ECLE surgery, the temporal expression patterns of E-cadherin (epithelial), α-SMA, fibronectin, and type I collagen (stromal) in the capsular bag were observed with or without involucretin (5 mM) treatment. At 0 hours, E-cadherin was highly expressed in both groups of anterior lens capsule LECs, while EMT markers were minimally expressed. Over time, EMT marker protein expression gradually increased in the control group, while it significantly decreased in the involucretin treatment group. E-cadherin expression in the anterior lens capsule of the control group gradually decreased over time, almost disappearing. In contrast, this decreasing trend was mitigated after involucretin injection into the anterior chamber. Western blot and qRT-PCR analyses confirmed that involucretin can inhibit EMT processes in the capsular bag after ECLE surgery. Figure 6 A- Figure 6 F).

[0093] HE staining results showed that costunolide could inhibit the proliferation and migration of anterior bursal epithelial cells after ECLE surgery. Figure 7 The modeling procedure involved preserving the lens capsule while removing the cortex and nucleus. Notably, only a thin layer of lens endothelial cells (LECs) was observed in the anterior capsule after modeling. By postoperative day 5, LECs in the control group proliferated and migrated from the equatorial region to the posterior capsule, with a significant increase in cell number in the posterior capsule region adjacent to the pupil. In contrast, the posterior capsule in the costunolide treatment group remained largely transparent, with a significantly reduced cell number and no adhesion, indicating a significant reduction in the progression of polymorphic keratitis (PCO).

[0094] 2. Costus lactone inhibits the inflammatory response after extracapsular lens extraction.

[0095] Previous studies (Fan et al., 2024b; Jiang et al., 2018) have shown that LECs produce a large number of pro-inflammatory factors after ECLE surgery, and inflammation is a major cause of postoperative lens fibrosis. Four key inflammatory biomarkers—CXCL1, COX-2, G-CSF, and S100A9—were identified, and they changed significantly within 24 hours postoperatively. These biomarkers were subsequently used as indicators to assess the efficacy of anti-inflammatory drugs. The role of costunolactone in the postoperative inflammatory response of ECLE is as follows... Figure 8-9 As shown. Immunofluorescence staining ( Figure 8 The results showed that the levels of inflammatory factors in LECs were low immediately after ECLE surgery, but increased significantly at 24 hours. Intra-atrial injection of costunolide effectively suppressed the upward trend of the inflammatory response. Furthermore, qRT-PCR results ( Figure 9The results showed that inflammatory factor expression was significantly reduced in the cysts treated with costus lactone 24 hours after surgery, confirming the anti-inflammatory effect of costus lactone.

[0096] 3. Aucklandia lactone attenuates TGF-β2-induced proliferation, migration, and EMT of LECs in vitro.

[0097] The inhibitory effect of costunolide on TGF-β2-driven LECs response, such as Figure 10 As shown. First, to determine the optimal drug concentration, the effect of different concentrations of costunolide on the activity of LECs was investigated using a cell viability assay (CCK-8 assay). Figure 10 A). The results showed that a concentration of 5 μmol / L had no adverse effect on cell viability. Therefore, to further investigate the inhibitory effect of costunolide on the EMT process in LECs, 5 μmol / L was selected as the working concentration of costunolide in subsequent experiments. qRT-PCR ( Figure 10 B) and Western blotting experiments ( Figure 10 C) This study confirmed that stimulation with 10 ng / ml TGF-β2 upregulated the expression of EMT protein markers (α-SMA, Col I, FN) in LECs. Conversely, in the group pretreated with costunolide, the expression levels of these markers were decreased, a finding also confirmed by immunofluorescence assays. Figure 11 ).

[0098] The migration ability of LECs was assessed using cell scratch (horizontal migration) and Transwell (vertical migration) assays. Figure 10 (DG). Compared with the control group, TGF-β2 stimulation significantly enhanced the horizontal and vertical migration abilities of LECs, as evidenced by increased scratch healing rate and increased number of transwell cells, respectively. However, costunolide treatment effectively inhibited this TGF-β2-induced pro-migration effect, resulting in a significant reduction in cell invasion and scratch repair capabilities. These results collectively indicate that costunolide can alleviate the TGF-β2-induced pro-migration phenotype of LECs.

[0099] 4. Transcriptome analysis of genes related to costunolide regulation after ECLE surgery in mice.

[0100] To further elucidate the mechanism by which costunolide inhibits EMT in LECs, lens capsules from different experimental groups were collected for transcriptome analysis. Five experimental groups were established: three groups received ECLE combined with saline injection, with samples collected at 0, 24, and 5 days (NS-0h, NS-24h, NS-5d); two groups received ECLE combined with costunolide injection, with samples collected at 24 hours and 5 days (MCL-24h, MCL-5d). Principal component analysis (PCA) was performed based on the FPKM values ​​of each group's genes. Each group contained three biological replicates, with good intra-group reproducibility, confirming the reliability of the sequencing data. Figure 12 The results show (). Figure 13 According to the screening criteria (padj ≤ 0.05 and |log2 (fold change)| ≥ 1), compared with the control group (NS-0h), a total of 4035 differentially expressed genes (DEGs) were upregulated and 964 genes were downregulated in the cyst tissue of the NS-24h group. KEGG pathway enrichment analysis of DEGs (…) Figure 14 The results showed that these genes were mainly enriched in the PI3K-Akt signaling pathway and the MAPK signaling pathway. Furthermore, analysis of the NS-5d group identified 3905 upregulated DEGs and 868 downregulated genes. Figure 15 B). KEGG enrichment analysis of these DEGs ( Figure 16 The results indicate that they are significantly enriched in pathways such as cytokine-cytokine receptor interaction, cell adhesion molecules, and ECM-receptor interaction.

[0101] Transcriptome comparative analysis was performed on the cyst tissue at 0 hours, 24 hours and 5 days after ECLE surgery. Figure 15 A shows a heatmap of 13 representative DEGs associated with EMT and inflammation. Notably, inflammation-related genes such as CXCL1, LCN2, G-CSF, and S100A9 showed the highest expression levels at 24 hours post-surgery. Conversely, the expression of EMT-related markers (including TGF-β, Col1a1, FN1, and tendinogen TNC) peaked on day 5 post-surgery. Finally, using p ≤ 0.05 and |log2 (fold change)| ≥ 1 as criteria, 347 DEGs were identified by comparing the MCL-24h and NS-24h groups. Figure 18 Overlap analysis of these DEGs with those of the NS-24h group vs. the NS-0h group revealed 206 common DEGs. Figure 17 A). Among them, 23 genes that were upregulated in the NS-24h group were downregulated after MCL treatment ( Figure 17(B) suggests that these may be potential molecular targets for the action of costunolide. Notably, compared to the NS-24h group, the MMP8 gene was most significantly downregulated in the costunolide treatment group (log2 fold change = -3.289). Figure 18 These findings lead to the hypothesis in this application that costunolide inhibits the EMT process of LCEs by downregulating the expression of MMP8.

[0102] 5. To verify the hypothesis that costunolide inhibits EMT by regulating MMP8 expression, this application performed qRT-PCR and Western blot analysis on the cyst tissue collected 24 hours after surgery. The results showed that costunolide treatment significantly reduced the protein expression and mRNA level of MMP8. Figure 19 A, B, C). Consistent results were observed in in vitro experiments, with costunolide effectively reducing MMP8 expression at both the protein and transcriptional levels. Notably, costunolide also attenuated TGF-β2-induced MMP8 upregulation ( Figure 19 D, E, F). This application incorporated M8I (an MMP8 inhibitor) into an in vitro model to further confirm the role of MMP8. Using the same cell viability assay, the maximum non-toxic concentration of M8I was determined to be 100 mmol / L. Figure 19 G). Under otherwise unchanged conditions, costunolide and M8I were administered simultaneously. Western blot analysis showed that, under TGF-β2 induction, the combined use of M8I was more effective than costunolide alone in inhibiting the expression and level of EMT marker proteins. Figure 19 H, I, J, K). Co-immunofluorescence staining of α-SMA and MMP8 also yielded consistent results. Figure 20 As shown, TGF-β2 induction significantly enhanced the expression of α-SMA and MMP8 in the cytoplasm, while costunolide treatment reduced their expression, and the combination of costunolide and M8I had a stronger inhibitory effect. These results collectively indicate that costunolide at least partially attenuates the EMT process by downregulating MMP8.

[0103] 4. Discussion

[0104] Advances in phacoemulsification and femtosecond laser-assisted techniques have made cataract surgery a highly efficient and safe ophthalmic procedure. However, the integrity of the blood-aqueous humor barrier can still be compromised during the procedure, accompanied by immune cell infiltration. Although the lens has historically been considered avascular tissue, research by Menko et al. has demonstrated that tissue-resident immune cells are a conserved feature of the lens epithelium in humans, mice, and chickens. These immune cells are activated during surgical stress, such as cataract surgery, and function as key regulators of wound healing responses, including the repair of fibrotic tissue. Further evidence indicates that multiple immune response genes are significantly upregulated in post-cataract surgery LECs compared to non-cataract controls. Disruption of the blood-aqueous humor barrier and immune cell infiltration are major triggers for the release of acute and subacute inflammatory cytokines.

[0105] Lens injury immediately triggers the production of pro-inflammatory cytokines in lens-associated capsular fibroblasts (LECs), leading to a chronic inflammatory response that persists for years after cataract surgery. Six hours after ECLE, significant upregulation of inflammatory factors was observed at the mRNA level, including IL19 upregulated 754-fold, COX2 upregulated 37-fold, and Csf3 upregulated 233-fold. In contrast, the mRNA levels of many fibrosis markers (such as α-SMA upregulated only 1.48-fold, and FN even downregulated) were not significantly elevated in LECs at this early time point. This indicates that the inflammatory response in LECs precedes the fibrotic response following lens injury. Furthermore, this inflammatory response may promote the formation of capsular bag-associated myofibroblasts, a characteristic feature of fibrotic PCO. Subsequently, after 24 hours of culture, LECs in the capsular bag tissue showed differential expression of EMT-related genes, including the classic lens EMT markers α-SMA and TNC. Consistent with this timeline, the measurements in this application show that inflammatory mediators, including CXCL1, G-CSF, and S100A9, peaked 24 hours after ECLE surgery and decreased on day 5; while EMT-related markers such as FN, Col I, TNC, and TGF-β began to rise at 24 hours and peaked on day 5. The results of this application elucidate the dynamic progression of LECs after cataract surgery: beginning with an early inflammatory phase, subsequently evolving into a fibrotic response. This process is driven by the migration of LECs from the anterior capsule to the posterior capsule, a key event in PCO (Potential Occurrence of Cellular Collapse). Although intraocular lenses (IOLs) employ a rectangular edge design and hydrophobic materials to inhibit LEC migration, the high migratory capacity of residual cells still allows them to reach the posterior capsule. Furthermore, genes related to cell migration are significantly upregulated in LECs after cataract surgery, and migration-related pathways are highly enriched. The experimental results of this application also confirm that residual LECs after ECLE surgery exhibit strong migratory capacity, completely covering the posterior capsule within 5 days post-surgery.

[0106] Current treatment strategies for PCO mainly focus on pharmacodynamics, gene therapy, and intraocular lens surface modification. Aucklandia lactone, a natural sesquiterpene lactone extracted from plants in the Asteraceae family, has been extensively studied and has demonstrated anti-inflammatory and anti-fibrotic properties. This application reports for the first time the efficacy of aucklandia lactone in slowing the progression of PCO. The optimal management goal for PCO is to achieve definitive prevention during the initial cataract surgery, thereby avoiding the need for secondary interventions, including repeat surgeries. Therefore, this application describes intra-atrial injection of aucklandia lactone immediately after ECLE. A three-month observational study demonstrated the effectiveness of intra-atrial injection of anti-VEGF drugs in inhibiting angiogenesis in pediatric vitreoretinal diseases. Furthermore, a large-scale meta-analysis involving 123,819 eyes of eight studies confirmed that intra-atrial injection of moxifloxacin effectively prevents post-cataract endophthalmitis. Therefore, intra-atrial injection is a feasible intervention strategy. Evaluation of corneal and retinal responses after administration confirmed that aucklandia lactone was well-tolerated, with no adverse reactions detected. However, drug loss due to aqueous humor circulation must be considered. One limitation of this study is the lack of a reliable method to quantify the concentration of costunolide in the anterior chamber, which warrants further investigation.

[0107] Having confirmed the efficacy of costunolide in reducing inflammatory response (PCO), this application focuses on its mechanism of action. Notably, among differentially expressed genes, MMP8 was found to be the most significantly downregulated target in response to costunolide treatment, indicating its central role in this process. MMP8, also known as neutrophil collagenase, is an important member of the matrix metalloproteinase family. Traditionally, MMP8 is thought to be primarily secreted by activated neutrophils and rapidly released upon neutrophil activation to ensure immediate availability at the site of inflammation. In a previous study (Jiang et al., 2018), CD11b and LY-6G (recognized neutrophil markers) positive cells were detected in the lens capsule as early as 18 hours after ECLE surgery. The presence of these cells increased continuously from 18 hours to 3 days post-surgery, remained at high levels on days 4 and 5, and significantly decreased by day 10. This temporal pattern of neutrophil infiltration provides evidence for the observed upregulation of MMP8 24 hours post-surgery. Several studies have reported the role of MMP8 in regulating the inflammatory response. Specifically, MMP8 is upregulated in LPS-stimulated BV2 microglia and primary cultured microglia, and mediates microglia activation by modulating TNF-α activity. Furthermore, a study (Wang et al., 2024) demonstrated that administration of M8I reduces microglia and astrocyte activation while alleviating oxidative stress in astrocytes. Consistently, elevated MMP8 expression is associated with the early inflammatory phase following spinal cord injury and leads to decreased expression of tight junction proteins in endothelial cells. Inhibition of MMP8 significantly reduces spinal cord injury-induced inflammation, blood-spinal barrier disruption, and cell damage. In addition to promoting hepatocellular carcinoma invasion and metastasis by inducing EMT via the PI3K / Akt / Rac1 pathway in synergy with TGF-β1, MMP8 has also been shown to enhance the migration and proliferation of vascular smooth muscle cells (VSMCs). Both in vitro and in vivo results confirmed that MMP8 expression was upregulated in the model group, while its expression was inhibited by treatment with costunolide. Notably, a synergistic effect was observed when costunolide was used in combination with M8I, significantly enhancing the inhibitory effect on EMT markers. In summary, this study is the first to systematically explore the efficacy and molecular mechanism of the natural compound costunolide in the prevention and treatment of PCO. Figure 21 The results of this application demonstrate that costunolide effectively reduces bag opacity after ECLE surgery, exhibiting significant anti-inflammatory and anti-fibrotic effects in both in vitro and in vivo models. Mechanistic studies identified MMP8 as a key downstream effector of costunolide, confirming that costunolide inhibits MMP8 expression, thereby blocking the transformation of LECs into mesenchymal cells and subsequent fibrosis, laying a solid foundation for developing targeted therapies for this common surgical complication.

[0108] The method of this invention has been described through preferred embodiments. Those skilled in the art will readily be able to modify or appropriately alter and combine the methods and applications described herein within the scope, spirit, and context of this invention to implement and apply the technology of this invention. Those skilled in the art can refer to the content herein to appropriately improve process parameters. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included within the scope of this invention.

Claims

1. Application of costunolide in the preparation of drugs for preventing posterior capsule opacification; The drug inhibits the expression of matrix metalloproteinase 8, thereby blocking the transformation of lens epithelial cells into mesenchymal cells and the subsequent fibrotic process.

2. The application according to claim 1, characterized in that, The drug also includes a matrix metalloproteinase 8 inhibitor.

3. The application according to claim 1, characterized in that, The drug is administered via intracavitary injection.

4. The application according to claim 1, characterized in that, The posterior capsule opacification is a secondary cataract following extracapsular lens extraction.

5. The application according to claim 4, characterized in that, The drug is administered via intraocular injection immediately after extracapsular lens extraction.

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