阅读说明：本技术 橙花叔醇在制备用于治疗真菌性角膜炎的药物中的应用 (Application of nerolidol in preparation of medicine for treating fungal keratitis ) 是由 车成业 杨桦 于 2019-11-26 设计创作，主要内容包括：本发明属于制药技术领域,公开了橙花叔醇在制备用于治疗真菌性角膜炎的药物中的应用。本发明首次提出,橙花叔醇能够抑制真菌生长,并且在体内和体外条件下对真菌性角膜炎具有治疗作用。在体内实验中,采用C57BL/6小鼠建立真菌性角膜炎模型,研究橙花叔醇对模型小鼠角膜临床评分、中性粒细胞募集、人氧化低密度脂蛋白受体1和白细胞介素1β表达的影响。在体外实验中,研究了不同浓度橙花叔醇对烟曲霉菌的生长具有抑制作用,同时采用烟曲霉菌刺激的人角膜上皮细胞模型验证了橙花叔醇对LOX-1/IL-1β信号通路的影响。(The invention belongs to the technical field of pharmacy, and discloses application of nerolidol in preparation of a medicine for treating fungal keratitis, which is provided for the first time, the nerolidol can inhibit the growth of fungi and has a treatment effect on the fungal keratitis under in-vivo and in-vitro conditions.)

1. Application of nerolidol in preparing medicine for treating fungal keratitis is provided.

2. The use according to claim 1, wherein nerolidol inhibits corneal fungal growth.

3. Use according to claim 2, wherein the corneal fungus is Aspergillus fumigatus.

4. The use of claim 2, wherein nerolidol inhibits the LOX-1/IL-1 β signaling pathway in corneal tissue.

5. The use of claim 3, wherein the LOX-1/IL-1 β signaling pathway in corneal tissue is the LOX-1/IL-1 β signaling pathway in human corneal epithelial cells stimulated by aspergillus fumigatus.

6. The medicine for treating fungal keratitis is characterized by comprising nerolidol and pharmaceutically acceptable auxiliary materials.

Technical Field

The invention belongs to the technical field of pharmacy, and particularly relates to application of nerolidol in preparation of a medicine for treating fungal keratitis.

Background

Fungal Keratitis (FK) is an infectious eye disease caused by pathogenic fungi, can cause corneal ulceration and perforation, and is a highly blinding, intractable corneal disease. Common pathogenic bacteria include fusarium and aspergillus. In China, vegetative trauma to eyes is the main cause of diseases, so that aspergillus fumigatus related to plants becomes the most common pathogenic bacteria of fungal keratitis patients in China. In clinical work, patients have untimely visits, and common antifungal drugs have the defects of poor water solubility, poor corneal penetration and the like, so that the treatment effect is often poor, partial patients develop corneal perforation and even blindness, and it is very necessary to find a safe and effective drug for inhibiting corneal fungal infection.

After fungal infection of the cornea, mutual recognition of pattern recognition receptors in the cornea and pathogen-associated molecular patterns on the fungal surface initiates an innate immune response of the cornea to clear fungal pathogens. After the cornea innate immune response is initiated, a large number of neutrophils and chemokines accumulate, causing corneal edema and inflammatory infiltration, and even causing corneal ulceration and even perforation. Therefore, too strong an immune response damages corneal tissue while controlling extrinsic infection, making it difficult to restore transparency of the cornea, and causing severe damage to the patient's vision. Therefore, protection of the cornea from fungal infection and avoidance of an exaggerated inflammatory response by the cornea are key to the treatment of fungal keratitis.

Nerolidol is natural semi-sesquiterpene alcohol extracted from various flower and plant essential oils, has the advantages of being natural, easy to obtain and small in side effect, and has attracted attention in many industries in recent years.

Disclosure of Invention

The invention aims to provide a new pharmaceutical application of nerolidol.

In order to solve the technical problems, the embodiment of the invention provides application of nerolidol in preparing a medicament for treating fungal keratitis.

In the pharmaceutical application provided by the invention, nerolidol is used for inhibiting the growth of corneal fungi, so that the treatment effect of inhibiting the inflammatory reaction degree of the cornea is achieved. Wherein, the corneal fungus is aspergillus fumigatus.

In the pharmaceutical application provided by the invention, the nerolidol inhibits a LOX-1/IL-1 β signal pathway in corneal tissue, more specifically, the LOX-1/IL-1 β signal pathway in the corneal tissue is a LOX-1/IL-1 β signal pathway in human corneal epithelial cells stimulated by aspergillus fumigatus.

The embodiment of the invention also provides a medicine for treating fungal keratitis, which comprises nerolidol and pharmaceutically acceptable auxiliary materials.

The invention provides application of nerolidol and aspergillus fumigatus spores in preparation of a medicine for treating fungal keratitis for the first time, the nerolidol and the aspergillus fumigatus spores with different concentrations are cultured for 24 hours to research the inhibition effect of the nerolidol on the aspergillus fumigatus, in vivo research, a C57BL/6 mouse is adopted to establish a fungal keratitis model, and the clinical evaluation of the nerolidol on the cornea of the model mouse, the recruitment of neutrophils and the influence of an LOX-1/IL-1 β signal path are researched.

Drawings

FIG. 1 is a graph showing the results of culturing Aspergillus fumigatus spores in example 1 in nerolidol at various concentrations for 24 hours in optical density values;

FIG. 2 is a graph showing the results of fungal cell wall staining in nerolidol treatment groups at different concentrations in example 1;

FIG. 3 is a graph of the comparison of the corneas of PBS-treated infected group and nerolidol-treated infected group under a slit lamp microscope after 3 days of establishment of the mouse model of fungal keratitis in example 2;

FIG. 4 is a graph showing the results of clinical scores of PBS-treated infected group and nerolidol-treated infected group 3 days after the fungal keratitis mouse model in example 2 was established;

FIG. 5 is a fluorescent plot of PBS-treated and nerolidol-treated infected groups using immunofluorescence techniques to detect inflammatory cell recruitment 2 days after the fungal keratitis mouse model in example 2 was established;

FIG. 6 shows the myeloperoxidase activity scores of the PBS-treated infected group and the nerolidol-treated infected group 2 days after the fungal keratitis mouse model in example 2 was established;

FIG. 7 is a graph showing the results of testing the effect of nerolidol on LOX-1mRNA expression in the cornea of a model mouse with fungal keratitis using the polymerase chain reaction in example 2;

FIG. 8 is a graph showing the results of Western blotting for the effect of nerolidol on LOX-1 protein secretion in the cornea of a mouse model of fungal keratitis in example 2;

FIG. 9 is a graph showing the results of testing the effect of nerolidol on the expression of IL-1 β mRNA in the cornea of a mouse model of fungal keratitis using the polymerase chain reaction in example 2;

FIG. 10 is a graph showing the results of Western blotting for the effect of nerolidol on the secretion of IL-1 β protein in the cornea of a mouse model of fungal keratitis in example 2;

FIG. 11 is a graph showing the results of detecting the effect of nerolidol on LOX-1mRNA expression in human corneal epithelial cells stimulated with fungi using the polymerase chain reaction in example 3;

FIG. 12 is a graph showing the results of Western blotting to examine the effect of nerolidol on LOX-1 protein secretion in human corneal epithelial cells stimulated with fungi in example 3;

FIG. 13 is a graph showing the results of testing the effect of nerolidol on the expression of IL-1 β mRNA in human corneal epithelial cells stimulated with fungi using the polymerase chain reaction in example 3;

FIG. 14 is a graph showing the results of Western blotting to examine the effect of nerolidol on the secretion of IL-1 β protein in human corneal epithelial cells stimulated by fungi in example 3.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be described in detail below. However, it will be appreciated by those of ordinary skill in the art that numerous technical details are set forth in order to provide a better understanding of the present application in various embodiments of the present invention. However, the technical solutions claimed in the claims of the present application can be implemented without these technical details and with various changes and modifications based on the following embodiments.

The invention mainly discusses the inhibition effect of nerolidol on the growth of aspergillus fumigatus and the treatment effect on fungal keratitis under in vivo and in vitro conditions in an in vivo research, a C57BL/6 mouse is adopted to establish a fungal keratitis model, and the influence of nerolidol on clinical corneal scores, neutrophil recruitment and an LOX-1/IL-1 β signal path of the model mouse is researched.

The experimental protocol and results established for the embodiments of the present invention are as follows:

dissolving conidia in Sasa culture solution to obtain a solution with a concentration of 5 × 10³The cfu/ml mixed solution is cultured for 24 hours in an incubator at 37 ℃ with nerolidol of different concentrations. The optical density at 460nm was measured. Subsequently, the hyphal cell wall was fluorescently stained. The results showed that the concentration of nerolidol at 100. mu.M significantly inhibited the growth of Aspergillus fumigatus and at 800. mu.M completely inhibited the germination of Aspergillus fumigatus spores, as compared to the control group.

In vivo experiments, a model of a mouse with fungal keratitis was established by intrastromal injection, different groups of the model mice were intervened by subconjunctival injection of nerolidol or an equal amount of PBS, the mouse cornea was observed daily using a slit lamp microscope and photographs were taken, the mouse eye and cornea were collected the next day, recruitment of corneal neutrophils was examined by immunofluorescence and Myeloperoxidase (MPO) assays, the mouse cornea was collected the 3 rd day, mRNA expression of LOX-1 and IL-1 β in the cornea was examined by polymerase chain reaction assays, and levels of LOX-1 and IL-1 β in the cornea were examined by Western immunoblotting assays.

In vitro experiments, the anti-inflammation mechanism of in vivo studies was verified by using human corneal epithelial cells.fumigatus conidia stimulate human corneal epithelial cells, and then different groups are intervened by nerolidol or PBS, cells are collected at 8 hours or 16 hours, the cells are collected at 8 hours and used for detecting mRNA expression of LOX-1 and IL-1 β in the human corneal epithelial cells through a polymerase chain reaction experiment, the cells are collected at 16 hours and used for detecting protein levels of LOX-1 and IL-1 β in the human corneal epithelial cells through a Western blot experiment, the results are consistent with the results of a mouse keratitis model, and compared with PBS control, the nerolidol intervention can inhibit LOX-1/IL-1 β in the aspergillus fumigatus stimulation group.