Application of isoimperatorin in preparation of medicine for treating fungal keratitis
Isorhynchonine addresses the problem of poor efficacy of existing antifungal drugs by inhibiting neutrophil recruitment and regulating signaling pathways in fungal keratitis, thus achieving effective treatment for fungal keratitis.
Patent Information
- Application Number
- CN202511144525.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-15
- Publication Date
- 2025-10-28
AI Technical Summary
Existing antifungal drugs have limited efficacy in treating fungal keratitis, especially due to poor matrix permeability and the rising prevalence of drug-resistant strains, resulting in poor treatment outcomes. Furthermore, there is a lack of effective topical antifungal eye drops.
Using isoimperatorin as the active ingredient, a drug for treating fungal keratitis was prepared by inhibiting the recruitment of fungal-stimulated mouse corneal neutrophils, regulating the TLR4/MyD88/IKK/NF-κB signaling pathway, inhibiting cell apoptosis, and promoting macrophage polarization.
It effectively reduces the degree of corneal inflammation, decreases neutrophil recruitment, inhibits IL-1β expression, inhibits the TLR4/MyD88/IKK/NF-κB signaling pathway, inhibits cell apoptosis, and promotes macrophage polarization from M1 phenotype to M2 phenotype, thus achieving the effect of treating fungal keratitis.
Smart Images
Figure CN120837491A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of isoimperatorin in the preparation of drugs for treating fungal keratitis. Background Technology
[0002] Fungal keratitis (FK) is a common corneal infection that frequently leads to blindness, particularly in developing countries. It primarily affects young male agricultural workers engaged in outdoor activities. Epidemiological estimates place the global annual incidence at at least 1.05 million cases, with geospatial analysis showing a disproportionate number of cases in Asia and Africa, recording 84,143–115,697 cases of vision loss. FK patients often experience severe infection and inflammation, leading to corneal opacity and intense eye pain. Despite advances in biotechnology, the range of available antifungal agents remains limited, primarily including polyenes, triazoles, echinocandins, and pyrimidine analogs, which can be used alone or in combination with systemic therapy. No new topical antifungal eye drops have been approved since the introduction of natamycin in the 1960s. However, the effectiveness of topical natamycin in 5% of cases is limited by its poor stromal permeability. The rising prevalence of drug-resistant Fusarium strains and increased biofilm formation exacerbate this problem.
[0003] The pathogenesis of fungal keratitis (FK) involves a complex interaction between host immune dysregulation and fungal invasion. Following corneal colonization, the innate immune system is rapidly activated, with neutrophils playing a crucial role in the early inflammatory response. These cells eliminate pathogens by releasing reactive oxygen species and elastases, but overactivation can lead to corneal stromal protein hydrolysis and barrier dysfunction. Simultaneously, macrophages undergo dynamic phenotypic changes—initial M1 polarization enhances inflammation through pro-inflammatory cytokines, while subsequent M2 polarization promotes tissue repair through the secretion of interleukin-10 (IL-10) and transforming growth factor-β (TGF-β). This imbalance also induces a caspase-dependent apoptosis cascade in corneal epithelial cells. Abnormal mitochondrial apoptotic signaling not only exacerbates ulceration but also perpetuates immune activation by releasing damage-associated molecular patterns (DAMPs), establishing a self-sustaining cycle of inflammation and apoptosis.
[0004] Isohippeastrin is a naturally occurring coumarin compound and one of the active components of coumarins. Studies have found that isohippeastrin possesses anti-inflammatory, analgesic, antitumor, and antiviral pharmacological effects. It can induce apoptosis in cancer cells by regulating the MAPK / ERK1 / 2 signaling pathway. Furthermore, isohippeastrin has been found to reduce oxidative stress in periodontal ligament cells, inhibit the secretion of pro-inflammatory factors in periodontal ligament cells, and activate the ERK1 / 2 and nuclear factor κB pathways. However, the anti-inflammatory mechanism of isohippeastrin remains unclear, and its potential applications in follicular keratosis (FK) have not been systematically investigated. Summary of the Invention
[0005] The purpose of this invention is to provide new pharmaceutical uses for isoimperatorin.
[0006] To address the aforementioned technical problems, embodiments of the present invention provide the use of isoimperatorin in the preparation of a medicament for treating fungal keratitis.
[0007] In the pharmaceutical applications provided by this invention, isoimperatorin is used to inhibit the recruitment and secretion of IL-1β by fungal-stimulated mouse corneal neutrophils, thereby reducing the clinical score of the mouse cornea and achieving a therapeutic effect of inhibiting the degree of corneal inflammation. The corneal fungus is *Aspergillus fumigatus*, and the mouse is a C57BL / 6 mouse.
[0008] In the pharmaceutical applications provided by the present invention, the isoimperatorin inhibits the TLR4 / MyD88 / IKK / NF-κB signaling pathway in the cornea of mice stimulated by Aspergillus fumigatus.
[0009] In the pharmaceutical applications provided by this invention, the isoimperatorin inhibits apoptosis in mouse corneas stimulated by Aspergillus fumigatus and promotes macrophage polarization.
[0010] Embodiments of the present invention also provide a medicament for treating fungal keratitis, the medicament comprising isoimperatorin and pharmaceutically acceptable excipients.
[0011] This invention proposes for the first time the application of isoimperatorin in the preparation of drugs for treating fungal keratitis. A fungal keratitis model was established using C57BL / 6 mice. The effects of isoimperatorin on corneal clinical scores, neutrophil recruitment, and interleukin-1β expression in the model mice were investigated. The effects of isoimperatorin on the TLR4 / MyD88 / IKK / NF-κB signaling pathway were also verified, as well as its effects on apoptosis and macrophage polarization. These experimental results confirm that isoimperatorin achieves a therapeutic effect against fungal keratitis. Attached Figure Description
[0012] Figure 1These are comparison images of the corneas of mice in the DMSO-treated and isoimperatorin-treated infection groups under a slit-lamp microscope 1, 3, and 5 days after the establishment of the fungal keratitis mouse model.
[0013] Figure 2 The image shows the clinical scores of the DMSO-treated and isoimperatorin-treated infection groups 1, 3, and 5 days after the establishment of the fungal keratitis mouse model.
[0014] Figure 3 Three days after the establishment of the fungal keratitis mouse model, the fluorescence images of inflammatory cell recruitment in the DMSO-treated and isoimperatorin-treated infection groups were detected using immunofluorescence technology.
[0015] Figure 4 This is a figure showing the results of Western blotting analysis to detect the effect of isoimperatorin on IL-1β protein secretion in the cornea of a mouse model of fungal keratitis.
[0016] Figure 5 The figure shows the results of Western blotting analysis to detect the effect of isoimperatorin on TLR4 / MyD88 / IKK / NF-κB signaling pathway proteins in the cornea of a mouse model of fungal keratitis.
[0017] Figure 6 This is a fluorescence image showing the recruitment of key proteins in the TLR4 and MyD88 signaling pathways in the cornea of a mouse model of fungal keratitis, detected using immunofluorescence technology.
[0018] Figure 7 This is a figure showing the results of flow cytometry analysis to detect the effect of isoimperatorin on corneal cell apoptosis in a mouse model of fungal keratitis.
[0019] Figure 8 This is a graph showing the results of flow cytometry analysis to detect the effect of isoimperatorin on the polarization of corneal macrophages in a mouse model of fungal keratitis.
[0020] Figure 9 The images show fluorescence images of macrophage recruitment detected using immunofluorescence technology in the DMSO-treated and isoimperatorin-treated infection groups three days after the establishment of the fungal keratitis mouse model. Detailed Implementation
[0021] To make the objectives, technical solutions, and advantages of this invention clearer, the various embodiments of this invention will be described in detail below. However, those skilled in the art will understand that many technical details have been provided in the various embodiments of this invention to facilitate a better understanding of this application. However, the technical solutions claimed in the claims of this application can be implemented even without these technical details and with various variations and modifications based on the following embodiments.
[0022] Imperatorin is a naturally occurring coumarin compound with certain medicinal potential. This invention primarily explores the following: A fungal keratitis model was established using C57BL / 6 mice. The effects of imperatorin on corneal clinical scores, neutrophil recruitment, and interleukin-1β expression in the model mice were investigated. The effects of imperatorin on the TLR4 / MyD88 / IKK / NF-κB signaling pathway were also verified. Furthermore, the effects of imperatorin on apoptosis and macrophage polarization were also verified.
[0023] The experimental scheme and results established for the embodiments of the present invention are as follows:
[0024] A mouse model of fungal keratitis was established using intrastromal injection. Different groups of mice were treated with subconjunctival injections of either isoimperatorin or an equal volume of DMSO. The mouse corneas were observed and photographed daily using a slit-lamp microscope. On day 3, mouse eyes and corneas were collected, and neutrophil recruitment was detected using immunofluorescence. Corneal tissue was also collected on day 3, and IL-1β protein levels were detected using Western blotting. Results showed that compared to the DMSO control group, the isoimperatorin-treated fungal infection group exhibited reduced corneal inflammation, decreased neutrophil recruitment, and lower IL-1β protein levels.
[0025] The anti-inflammatory mechanism was then validated in vivo. Different groups of model mice were treated with subconjunctival injection of either isoimperatorin or an equal volume of DMSO. On day 3, mouse eyes and corneas were collected, and the recruitment of key proteins in the corneal TLR4 and MyD88 signaling pathways was detected using immunofluorescence. On day 3, mouse corneas were also collected, and the protein levels of the TLR4 / MyD88 / IKK / NF-κB signaling pathways in the cornea were detected using Western blotting. The results showed that, compared with the DMSO control group, the isoimperatorin-treated fungal infection group had reduced corneal TLR4 / MyD88 / IKK / NF-κB signaling pathway protein levels.
[0026] Mice were treated with subconjunctival injection of either isoimperatorin or an equal volume of DMSO in different groups. On day 3, mouse eyes and corneas were collected. Flow cytometry was used to detect the effects of isoimperatorin on corneal cell apoptosis and macrophage polarization in the fungal keratitis model mice. On day 3, mouse eyes and corneas were also collected, and immunofluorescence was used to detect corneal macrophage recruitment. Results showed that, compared with the DMSO control group, the isoimperatorin-treated fungal infection group exhibited inhibited corneal apoptosis, suppressed macrophage recruitment, and promoted macrophage polarization from the M1 phenotype to the M2 phenotype.
[0027] Example 1: Effects of isoimperatorin on corneal clinical score, neutrophil recruitment, and IL-1β signaling pathway in a mouse model of fungal keratitis.
[0028] 1. Experimental Materials
[0029] 1.1 Experimental reagents: Isobellocin (purchased from MCE Company)
[0030] 1.2 Experimental animals: C57BL / 6 mice (purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd.)
[0031] 1.3 Experimental fungi: Aspergillus fumigatus strain 3.0772 (China General Microbiological Culture Collection Center)
[0032] 2. Experimental Methods
[0033] 2.1 Model Mice
[0034] Eight-week-old healthy, clean-grade female C57BL / 6 mice were selected as research subjects. Before the experiment, slit-lamp examination was used to rule out eye diseases, and the right eye was selected as the experimental eye. All procedures performed on the experimental mice complied with the Chinese Ministry of Science and Technology's Guidelines on Humanized Treatment of Laboratory Animals (vGKFCZ-2006–398) and the principles and standards of the Association for Research in Vision and Ophthalmology (ARVO) regarding the use of animals in ophthalmological and vision research.
[0035] 2.2 Effects of isoimperatorin on corneal clinical score, neutrophil recruitment, and IL-1β signaling pathway in a mouse model of fungal keratitis
[0036] C57BL / 6 mice were randomly divided into four groups: a DMSO control group, a DMSO-treated fungal infection group, an isoimperatorin-treated group, and an isoimperatorin-treated fungal infection group. After anesthetizing the mice with an intraperitoneal injection of 8% chloral hydrate, 2.5 μl of a 2.5 × 10⁻⁶ chloral solution was injected using a microsyringe. 6 A CFU / ml conidial suspension was injected into the corneal stroma of mice in the fungal infection group and the isoimperatorin-treated fungal infection group. Thirty minutes later, 5 μL of PBS was injected subconjunctivally into the control and fungal infection groups, while 5 μL of isoimperatorin (50 μg / ml) dissolved in DMSO was injected subconjunctivally into the isoimperatorin-treated and isoimperatorin-treated fungal infection groups. After modeling, the mouse cornea was observed and photographed daily using a slit-lamp microscope. On days 1, 2, and 3 after modeling, mice in each group were injected subconjunctivally with either DMSO or isoimperatorin. On day 3 after modeling, mice were sacrificed, and the eyeballs were harvested for immunofluorescence experiments, while the corneas were harvested for Western blotting analysis.
[0037] 3. Experimental Results
[0038] 3.1 Effect of isoimperatorin on corneal clinical scores in a mouse model of fungal keratitis
[0039] Figure 1 This is a comparison of the corneas of mice in the DMSO-treated and isoimperatorin-treated infection groups under a slit-lamp microscope 3 days after the establishment of the fungal keratitis mouse model. Figure 2 This is a graph showing the clinical scores of the DMSO-treated and isoimperatorin-treated infection groups three days after the establishment of the fungal keratitis mouse model.
[0040] like Figure 1 , 2 As shown, compared with the DMSO-treated infection group, the degree of corneal inflammation and clinical score of mice in the isoimperatorin-treated infection group were significantly reduced.
[0041] 3.2 Effects of isoimperatorin on the recruitment of inflammatory cells in the cornea of a mouse model of fungal keratitis
[0042] Figure 3 Three days after the establishment of the fungal keratitis mouse model, the fluorescence images of inflammatory cell recruitment in the DMSO-treated and isoimperatorin-treated infection groups were detected using immunofluorescence technology.
[0043] like Figure 3 As shown, compared with the DMSO-treated infection group, isoimperatorin treatment significantly reduced the number of neutrophils recruited in the cornea of mice with fungal keratitis 3 days after infection.
[0044] 3.3 Effects of isoimperatorin treatment on corneal IL-1β secretion in a mouse model of fungal keratitis
[0045] Figure 4 This figure shows the results of Western blotting analysis to detect the effect of isoimperatorin on IL-1β protein secretion in the cornea of a mouse model of fungal keratitis.
[0046] like Figure 4 As shown, Western blot experiments revealed that the protein level of IL-1β in the cornea of mice in the fungal infection group treated with isoimperatorin was significantly lower than that in the DMSO-treated infection group.
[0047] Example 2: Isorhynchin can inhibit the TLR4 / MyD88 / IKK / NF-κB signaling pathway in fungal-stimulated human corneal epithelial cells, inhibiting apoptosis and promoting macrophage polarization.
[0048] 1. Experimental Materials
[0049] 1.1 Experimental reagents: Isobellocin (purchased from MCE Company)
[0050] 1.2 Experimental animals: C57BL / 6 mice (purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd.)
[0051] 1.3 Experimental fungi: Aspergillus fumigatus strain 3.0772 (China General Microbiological Culture Collection Center)
[0052] 2. Experimental Methods
[0053] 2.1 Model Mice
[0054] Eight-week-old healthy, clean-grade female C57BL / 6 mice were selected as research subjects. Before the experiment, slit-lamp examination was used to rule out eye diseases, and the right eye was selected as the experimental eye. All procedures performed on the experimental mice complied with the Chinese Ministry of Science and Technology's Guidelines on Humanized Treatment of Laboratory Animals (vGKFCZ-2006–398) and the principles and standards of the Association for Research in Vision and Ophthalmology (ARVO) regarding the use of animals in ophthalmological and vision research.
[0055] 2.2 Isorhynchin can inhibit the TLR4 / MyD88 / IKK / NF-κB signaling pathway in human corneal epithelial cells stimulated by fungi, inhibiting apoptosis and promoting macrophage polarization.
[0056] C57BL / 6 mice were randomly divided into four groups: a DMSO control group, a DMSO-treated fungal infection group, an isoimperatorin-treated group, and an isoimperatorin-treated fungal infection group. After anesthetizing the mice with an intraperitoneal injection of 8% chloral hydrate, 2.5 μl of a 2.5 × 10⁻⁶ chloral solution was injected using a microsyringe. 6 A CFU / ml conidial suspension was injected into the corneal stroma of mice in the fungal infection group and the isoimperatorin-treated fungal infection group. Thirty minutes later, 5 μL of PBS was injected subconjunctivally into the control and fungal infection groups, while 5 μL of isoimperatorin (50 μg / ml) dissolved in DMSO was injected subconjunctivally into the isoimperatorin-treated and isoimperatorin-treated fungal infection groups. After modeling, the mouse cornea was observed and photographed daily using a slit-lamp microscope. On days 1, 2, and 3 after modeling, mice in each group were injected subconjunctivally with either DMSO or isoimperatorin. On day 3 after modeling, mice were sacrificed, and the eyeballs were harvested for immunofluorescence experiments, while the corneas were harvested for Western blotting and flow cytometry analysis.
[0057] 3. Experimental Results
[0058] 3.1 Effects of isoimperatorin treatment on corneal TLR4 / MyD88 / IKK / NF-κB signaling pathway proteins in a mouse model of fungal keratitis
[0059] Figure 5 The figure shows the results of detecting the effect of isoimperatorin on the secretion of TLR4 / MyD88 / IKK / NF-κB proteins in the cornea of a mouse model of fungal keratitis using Western blotting. Figure 6 Three days after the establishment of the fungal keratitis mouse model, the fluorescence images of the recruitment of key proteins in the TLR4 and MyD88 signaling pathways were detected using immunofluorescence technology in the DMSO-treated and isoimperatorin-treated infection groups.
[0060] like Figure 5 , Figure 6 As shown, Western blotting and immunofluorescence experiments revealed that the levels of TLR4 / MyD88 / IKK / NF-κB signaling pathway proteins in the cornea of mice treated with isoimperatorin were significantly lower than those in the DMSO-treated infection group.
[0061] 3.2 Isohexadecanol can inhibit corneal cell apoptosis in a mouse model of fungal keratitis.
[0062] Figure 7 The figure shows the results of flow cytometry analysis to detect the effect of isoimperatorin on corneal cell apoptosis and secretion in a mouse model of fungal keratitis.
[0063] like Figure 7 As shown, flow cytometry experiments revealed that the level of corneal cell apoptosis in mice infected with fungi treated with isoimperatorin was significantly lower than that in mice infected with DMSO.
[0064] 3.3 Isobellocin can inhibit the recruitment of corneal macrophages and promote macrophage polarization in a mouse model of fungal keratitis.
[0065] Figure 8 This is a graph showing the results of flow cytometry analysis to detect the effect of isoimperatorin on the polarization of corneal macrophages in a mouse model of fungal keratitis. Figure 9 The images show fluorescence images of macrophage recruitment detected by immunofluorescence technology in the DMSO-treated and isoimperatorin-treated infection groups 3 days after the establishment of the fungal keratitis mouse model.
[0066] like Figure 8 As shown, flow cytometry and immunofluorescence experiments indicated that the polarization level of corneal macrophages in mice infected with fungi treated with isoimperatorin was significantly higher than that in the DMSO-treated group. Figure 9 As shown, compared with the DMSO-treated infection group, isoimperatorin treatment significantly reduced the number of macrophages recruited in the cornea of mice with fungal keratitis 3 days after infection.
[0067] Those skilled in the art will understand that the above embodiments are specific examples of implementing the present invention, and in practical applications, various changes in form and detail may be made without departing from the spirit and scope of the present invention.
Claims
1. The application of isoimperatorin in the preparation of drugs for the treatment of fungal keratitis.
2. The application according to claim 1, characterized in that, The isoimperatorin inhibits the recruitment and secretion of IL-1β by fungal-stimulated mouse corneal neutrophils.
3. The application according to claim 2, characterized in that, The fungus is Aspergillus fumigatus, and the mouse is a C57BL / 6 mouse.
4. The application according to claim 1, characterized in that, The isoimperatorin inhibits the TLR4 / MyD88 / IKK / NF-κB signaling pathway in the cornea of mice stimulated by Aspergillus fumigatus.
5. The application according to claim 1, characterized in that, The isoimperatorin inhibits apoptosis in mouse corneas stimulated by Aspergillus fumigatus and promotes macrophage polarization.
6. A drug for treating fungal keratitis, characterized in that, It contains isoimperatorin and pharmaceutically acceptable excipients.