Application of sennoside A in preparation of medicine for treating fungal keratitis
By using sennoside A to inhibit the inflammatory response in a mouse model of fungal keratitis, the problems of drug resistance and excessive inflammation in fungal keratitis were solved, achieving effective treatment and reducing corneal inflammation and tissue damage.
Patent Information
- Application Number
- CN202511380759.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-25
- Publication Date
- 2025-11-11
AI Technical Summary
In existing technologies, treatments for fungal keratitis suffer from drug resistance issues, and excessive inflammatory responses can lead to irreversible vision damage, making the search for more effective treatments particularly important.
Sennoside A was used to inhibit the recruitment of neutrophils and macrophages and the secretion of IL-1β in the cornea of mice stimulated by fungi, thereby reducing corneal inflammation by inhibiting the Caspase-1/GSDMD signaling pathway.
It effectively inhibits the inflammatory response in a mouse model of fungal keratitis, reduces corneal clinical scores, decreases immune cell recruitment, promotes macrophage polarization, alleviates corneal tissue damage, and prevents irreversible visual impairment.
Smart Images
Figure CN120919149A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of sennoside A in the preparation of a drug for treating fungal keratitis. Background Technology
[0002] Fungal keratitis (FK) is a blinding corneal disease caused by direct infection of the corneal tissue by pathogenic fungi. It is a major cause of infectious corneal ulcers and even blindness, with a particularly high incidence in developing countries where agriculture is the primary activity. It is often associated with ocular trauma (especially plant-related injuries). Globally, filamentous fungi are the predominant causative agents of fungal keratitis, with Fusarium and Aspergillus being the most common. In recent years, with the widespread use of broad-spectrum antibiotics and corticosteroids, its global incidence has been on the rise, and it has developed resistance to conventional treatments. The pathological process of fungal keratitis is characterized by severe inflammation and corneal tissue destruction. Even with successful pathogen clearance, the excessive inflammation itself can lead to corneal opacity, neovascularization, and even perforation, causing irreversible visual impairment. Therefore, finding more effective treatments for fungal keratitis is crucial.
[0003] As an exposed ocular surface tissue, the cornea primarily relies on innate immunity for initial defense against fungal pathogens. Fungal pathogens possess pathogen-associated molecular patterns (MAMS), which can interact with pattern recognition receptors on host cells. When the cornea is subjected to mechanical damage or fungal infection, MAMS receptors can recognize these MAMS, initiating an intracellular signaling cascade. This process promotes inflammatory signal transduction and rapidly recruits key immune cells, including neutrophils and macrophages, to the site of infection. These inflammatory responses are central to the host's immune response; however, excessive inflammation can lead to the over-release of inflammatory factors, causing more severe corneal damage. Therefore, avoiding an excessively strong inflammatory response in the cornea is crucial for treating fungal keratitis.
[0004] Sennoside A is a dianthrone derivative found in senna leaves, a traditional Chinese medicine, and is often used as a stimulant laxative and a weight-loss health food. In recent years, sennoside A has been shown to have broad effects in various diseases, with studies demonstrating its anti-inflammatory, anti-tumor, antibacterial, and mitochondrial protective properties. However, its role in fungal keratitis remains unexplored. Summary of the Invention
[0005] The purpose of this invention is to provide new pharmaceutical uses for sennoside A.
[0006] To address the aforementioned technical problems, this invention provides the use of sennoside A in the preparation of a medicament for treating fungal keratitis.
[0007] In the pharmaceutical applications provided by this invention, sennoside A is used to inhibit the recruitment of neutrophils and macrophages and the secretion of IL-1β in the cornea of mice stimulated by fungi, thereby reducing the clinical score of the cornea in mice and achieving a therapeutic effect of alleviating the degree of corneal inflammation. The fungus is *Aspergillus fumigatus*, and the mice are C57BL / 6 mice.
[0008] In the pharmaceutical use provided by the present invention, sennoside A inhibits the Caspase-1 / GSDMD signaling pathway in the cornea of mice stimulated by Aspergillus fumigatus.
[0009] In the pharmaceutical applications provided by this invention, sennoside A inhibits the activity of neutrophils in the cornea of mice stimulated by Aspergillus fumigatus and promotes macrophage polarization.
[0010] The present invention also provides a medicament for treating fungal keratitis, the medicament comprising sennoside A and pharmaceutically acceptable excipients.
[0011] This invention proposes for the first time the application of sennoside A in the preparation of drugs for treating fungal keratitis. A fungal keratitis model was established using C57BL / 6 mice. The effects of sennoside A on corneal clinical scores, neutrophil recruitment, macrophage recruitment, and interleukin-1β expression in the model mice were investigated. The effects of sennoside A on the Caspase-1 / GSDMD signaling pathway, as well as its effects on neutrophil activity and macrophage polarization, were also verified. These experimental results confirm that sennoside A achieves a therapeutic effect against fungal keratitis. Attached Figure Description
[0012] Figure 1 shows a comparison of the corneas of mice in the DMSO-treated and sennoside A-treated infection groups under a slit-lamp microscope 1, 3, and 5 days after the establishment of the fungal keratitis mouse model. Figure 2 shows the clinical scores of the DMSO-treated and sennoside A-treated infection groups 1, 3, and 5 days after the establishment of the fungal keratitis mouse model. Figure 3 shows the results of Western blotting analysis of the effect of sennoside A on IL-1β protein secretion in the cornea of a mouse model of fungal keratitis. Figure 4 shows the results of Western blotting analysis to detect the effect of sennoside A on Caspase-1 / GSDMD signaling pathway proteins in the cornea of a mouse model of fungal keratitis. Figure 5 is a fluorescence diagram showing the recruitment of key proteins in the Caspase-1 / GSDMD signaling pathway in the cornea of a mouse model of fungal keratitis, detected by immunofluorescence technology. Figure 6This is a fluorescence image of neutrophil recruitment in the DMSO-treated and sennoside A-treated infection groups, detected by immunofluorescence technology one day after the establishment of the fungal keratitis mouse model. Figure 7 This is a fluorescence image of macrophage recruitment in the DMSO-treated and sennoside A-treated infection groups, detected by immunofluorescence technology 3 days after the establishment of the fungal keratitis mouse model. Figure 8 This is a figure showing the results of an enzyme-linked immunosorbent assay (ELISA) to detect the effect of sennoside A on myeloperoxidase in the cornea of a mouse model of fungal keratitis. Figure 9 This is a graph showing the results of flow cytometry analysis to detect the effect of sennoside A on the polarization of corneal macrophages in a mouse model of fungal keratitis. Detailed Implementation
[0013] 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.
[0014] Sennoside A is a single compound extracted from senna leaves and other traditional Chinese medicinal herbs, possessing certain medicinal potential. This invention primarily explores the following: A fungal keratitis model was established using C57BL / 6 mice. The effects of sennoside A on corneal clinical scores, neutrophil recruitment, macrophage recruitment, and interleukin-1β expression in the model mice were investigated. The effects of sennoside A on the Caspase-1 / GSDMD signaling pathway were also verified, along with its effects on neutrophil activity and macrophage polarization.
[0015] The experimental scheme and results established for the embodiments of the present invention are as follows: A mouse model of fungal keratitis was established using intrastromal corneal injection. Different groups of mice were treated with subconjunctival injections of sennoside A or an equal volume of DMSO. The mouse corneas were observed and photographed daily using a slit-lamp microscope. On days 1 and 3 post-infection, mouse eyes and corneas were collected. Immunofluorescence was used to detect the recruitment of corneal immune cells and to observe the effect of sennoside A treatment on neutrophil activity and macrophage polarization. On day 3, mouse corneas were collected, and Western blotting and immunofluorescence experiments were used to detect the protein levels of the Caspase-1 / GSDMD signaling pathway in the cornea.
[0016] The anti-inflammatory mechanism studied in vivo was validated. Different groups of model mice were intervened by subconjunctival injection of sennoside A or an equal volume of DMSO. Corneas were collected on day 3, and the protein levels of the Caspase-1 / GSDMD signaling pathway in the cornea were detected by Western blotting. Results showed that, compared with the DMSO control group, the corneal Caspase-1 / GSDMD signaling pathway protein levels were reduced in the sennoside A-treated fungal infection group.
[0017] To verify the effects of sennoside A on immune cells, different groups of model mice were treated by subconjunctival injection of sennoside A or an equal volume of DMSO. On day 1, mouse eyes and corneas were collected. Neutrophil recruitment was detected by immunofluorescence assay, and corneal myeloperoxidase levels, representing neutrophil activity, were detected by enzyme-linked immunosorbent assay (ELISA). On day 3, mouse eyes and corneas were collected again. Macrophage recruitment was detected by immunofluorescence assay, and the effect of sennoside A on corneal macrophage polarization in a mouse model of fungal keratitis was detected by flow cytometry. The results showed that, compared with the DMSO control group, the sennoside A-treated fungal infection group exhibited suppressed immune cell recruitment in the cornea, inhibited neutrophil activity, and promoted macrophage polarization from the M1 phenotype to the M2 phenotype.
[0018] Example 1: Sennoside A can inhibit the Caspase-1 / GSDMD signaling pathway in the cornea of mice stimulated by fungi, suppress the inflammatory response, and reduce the clinical corneal score of a mouse model of fungal keratitis.
[0019] 1. Experimental Materials 1.1 Experimental reagent: Sennoside A (purchased from MCE Company) 1.2 Experimental animals: C57BL / 6 mice (purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd.) 1.3 Experimental fungi: Aspergillus fumigatus strain 3.0772 (China General Microbiological Culture Collection Center) 2. Experimental Methods 2.1 Model Mice 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.
[0020] 2.2 Sennoside A can inhibit the Caspase-1 / GSDMD signaling pathway in the cornea of fungal-stimulated mice, suppress the inflammatory response, and reduce the clinical corneal score of a fungal keratitis model mouse.
[0021] C57BL / 6 mice were randomly divided into a DMSO control group, a DMSO-treated fungal infection group, a sennoside A-treated group, and a sennoside A-treated fungal infection group. After anesthetizing the mice with a mixture of ketamine (100 mg / kg) and toluenethiazide (15 mg / kg) administered intraperitoneally, 2.5 μl of a 2.5 × 10⁻⁶ solution was injected using a microsyringe. 6 A CFU / ml suspension of conidia was injected into the corneal stroma of mice in the fungal infection group and the sennoside A-treated fungal infection group. Thirty minutes later, 5 μL of PBS was injected subconjunctivally into mice in the control and fungal infection groups, while 5 μL of sennoside A dissolved in DMSO (100 μg / ml) was injected subconjunctivally into mice in the sennoside A-treated and sennoside A-treated fungal infection groups. After modeling, the mouse corneas were observed and photographed daily using a slit-lamp microscope. Each group of model mice was injected subconjunctivally with either DMSO or sennoside A daily as an intervention. Mice were sacrificed on day 3 after modeling, and corneas were harvested for Western blotting analysis.
[0022] 3. Experimental Results 3.1 Effect of sennoside A on corneal clinical scores in a mouse model of fungal keratitis; Figure 1 These are comparison images of the corneas of mice in the DMSO-treated and sennoside A-treated infection groups under a slit-lamp microscope 1, 3, and 5 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 sennoside A-treated infection groups 1, 3, and 5 days after the establishment of the fungal keratitis mouse model.
[0023] like Figure 1 , Figure 2 As shown, compared with the DMSO-treated infection group, the degree of corneal inflammation and clinical score of mice in the sennoside A-treated infection group were significantly reduced, indicating that it can effectively inhibit the inflammatory response of the cornea, alleviate the clinical manifestations of fungal keratitis, and accelerate the improvement of the disease.
[0024] 3.2 Effects of sennoside A treatment on corneal IL-1β secretion in a mouse model of fungal keratitis; Figure 3 This is a graph showing the results of Western blotting analysis to detect the effect of sennoside A on IL-1β protein secretion in the cornea of a mouse model of fungal keratitis.
[0025] like Figure 3As shown, Western blotting experiments revealed that the protein level of IL-1β in the cornea of mice treated with sennoside A was significantly lower than that in the DMSO-treated infection group. The significant reduction in the expression level of IL-1β, a key inflammatory factor, further confirms that sennoside A can inhibit the excessive release of inflammatory factors, thereby alleviating the degree of corneal inflammation.
[0026] 3.3 Effects of sennoside A treatment on corneal Caspase-1 / GSDMD signaling pathway proteins in a mouse model of fungal keratitis.
[0027] Figure 4 This figure shows the results of Western blotting analysis of the effect of sennoside A on the secretion of Caspase-1 / GSDMD signaling pathway-related proteins in the cornea of mice with fungal keratitis, 3 days after the establishment of the mouse model. Figure 5 This is a fluorescence image of the recruitment of GSDMD, a key protein in the signaling pathway, in the DMSO-treated and sennoside A-treated infection groups three days after the establishment of the fungal keratitis mouse model.
[0028] like Figure 4 , Figure 5 As shown, Western blotting and immunofluorescence experiments revealed that the levels of Caspase-1 / GSDMD signaling pathway proteins in the cornea of mice treated with sennoside A were significantly lower than those in the DMSO-treated infection group. This indicates that sennoside A can effectively control excessive inflammatory responses in the cornea by intervening in the pyroptosis pathway and reducing the activation of inflammation-related proteins.
[0029] Example 2: Sennoside A can inhibit the recruitment of immune cells in the cornea of mice infected with fungi, inhibit neutrophil activity and affect macrophage polarization.
[0030] 1. Experimental Materials 1.1 Experimental reagent: Sennoside A (purchased from MCE Company) 1.2 Experimental animals: C57BL / 6 mice (purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd.) 1.3 Experimental fungi: Aspergillus fumigatus strain 3.0772 (China General Microbiological Culture Collection Center) 2. Experimental Methods 2.1 Model Mice 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.
[0031] 2.2 Sennoside A can inhibit the recruitment of immune cells in the cornea of mice infected with fungi, inhibit neutrophil activity and affect macrophage polarization.
[0032] C57BL / 6 mice were randomly divided into a DMSO control group, a DMSO-treated fungal infection group, a sennoside A-treated group, and a sennoside A-treated fungal infection group. After anesthetizing the mice with a mixture of ketamine (100 mg / kg) and toluenethiazide (15 mg / kg) administered intraperitoneally, 2.5 μl of a 2.5 × 10⁻⁶ 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 sennoside A-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 sennoside A dissolved in DMSO (50 μg / ml) was injected subconjunctivally into the sennoside A-treated and sennoside A-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 sennoside A. Mice were sacrificed on days 1 and 3 after modeling; eyeballs were harvested for immunofluorescence experiments, and corneas were harvested for enzyme-linked immunosorbent assay (ELISA) and flow cytometry analysis.
[0033] 3. Experimental Results 3.1 Effect of sennoside A treatment on the recruitment of corneal immune cells in a mouse model of fungal keratitis; Figure 6 The images show fluorescence images of neutrophil recruitment detected by immunofluorescence technology in the DMSO-treated and sennoside A-treated infection groups one day after the establishment of the fungal keratitis mouse model. Figure 7 The images show fluorescence images of macrophage recruitment detected using immunofluorescence technology in the DMSO-treated and sennoside-6-treated infection groups three days after the establishment of the fungal keratitis mouse model.
[0034] like Figure 6 , Figure 7As shown, compared with the DMSO-treated infection group, sennoside A treatment can significantly reduce the number of inflammatory cells recruited in the cornea of mice with fungal keratitis after infection with Aspergillus fumigatus, indicating that it can effectively inhibit the excessive aggregation of immune cells at the infection site, thereby reducing the degree of inflammatory response and avoiding corneal tissue damage.
[0035] 3.2 Sennoside A can inhibit the activity of corneal neutrophils and affect macrophage polarization in a mouse model of fungal keratitis.
[0036] Figure 8 This is a graph showing the results of enzyme-linked immunosorbent assay (ELISA) to detect the effect of sennoside A on corneal myeloperoxidase in a mouse model of fungal keratitis one day after its establishment. Figure 9 This is a flow cytometry analysis of corneal macrophage polarization in a mouse model of fungal keratitis, three days after its establishment.
[0037] Myeloperoxidase is used as a biomarker for neutrophil activation. For example... Figure 8 As shown in Figure 9, the corneal myeloperoxidase level was decreased in mice treated with sennoside A for fungal infection, reflecting reduced neutrophil activity. Compared to the DMSO-treated infection group, the M2 polarization level of macrophages was increased in the sennoside A-treated group. Sennoside A not only reduces the recruitment of neutrophils and macrophages but also decreases neutrophil activity and promotes macrophage polarization towards the M2 anti-inflammatory phenotype. These effects collectively indicate that it can regulate the corneal immune response after fungal infection and inhibit excessive inflammation.
[0038] 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 use of sennoside A in the preparation of drugs for the treatment of fungal keratitis.
2. The application according to claim 1, characterized in that, Sennoside A inhibits the recruitment of neutrophils and macrophages and the secretion of IL-1β by fungi-stimulated mouse corneas.
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 3, characterized in that, Sennoside A inhibits the Caspase-1 / GSDMD signaling pathway in the cornea of mice stimulated by Aspergillus fumigatus.
5. The application according to claim 3, characterized in that, Sennoside A inhibits neutrophil activity in mouse cornea stimulated by Aspergillus fumigatus and promotes macrophage polarization.
6. A drug for treating fungal keratitis, characterized in that, It contains sennoside A and pharmaceutically acceptable excipients.