CN110840865A - Application of nerolidol in preparation of medicine for treating fungal keratitis - Google Patents

Abstract

The invention belongs to the technical field of pharmacy and discloses the application of nerolidol in preparing a medicine for treating fungal keratitis. The present invention proposes for the first time that nerolidol can inhibit the growth of fungi and has a therapeutic effect on fungal keratitis in vivo and in vitro. In vivo experiments, a fungal keratitis model was established in C57BL/6 mice, and the effects of nerolidol on the corneal clinical score, neutrophil recruitment, human oxidized low-density lipoprotein receptor 1 and interleukin of the model mice were studied. 1β expression. In vitro experiments, the inhibitory effect of different concentrations of nerolidol on the growth of Aspergillus fumigatus was studied, and the human corneal epithelial cell model stimulated by Aspergillus fumigatus was used to verify the effect of nerolidol on LOX-1/IL-1β signaling pathway effects.

Description

Application of nerolidol in the preparation of medicaments for the treatment of fungal keratitis

technical field

The invention belongs to the technical field of pharmacy, in particular to the application of nerolidol in the preparation of a medicament for treating fungal keratitis.

Background technique

Fungal keratitis (FK) is an infectious eye disease caused by pathogenic fungi, which can cause corneal ulcers and perforations. It is a corneal disease with high blindness and difficult to treat. Common pathogens include Fusarium and Aspergillus. In my country, ocular vegetative trauma is the main cause of disease, so Aspergillus fumigatus associated with plants has become the most common pathogen in Chinese patients with fungal keratitis. In clinical work, due to the lack of timely treatment by patients, and the shortcomings of common antifungal drugs such as poor water solubility and poor corneal penetration, the treatment effect is often poor, resulting in some patients developing corneal perforation or even blindness. Safe and effective drugs to inhibit corneal fungal infection are very necessary.

After fungal infection of the cornea, the mutual recognition of pattern recognition receptors in the cornea and pathogen-associated molecular patterns on the surface of the fungus initiates the innate immune response of the cornea to eliminate the fungal pathogen. After the corneal innate immune response is initiated, a large number of neutrophils and chemokines accumulate, leading to corneal edema and inflammatory infiltration, and even corneal ulcers and even perforation. Therefore, an excessively strong immune response causes damage to the corneal tissue while controlling the foreign infection, making it difficult for the cornea to restore transparency, and causing serious damage to the patient's vision. Therefore, protecting the cornea against fungal infection and avoiding excessive corneal inflammation is the key to the treatment of fungal keratitis.

Nerolidol is a natural hemisesterpene alcohol extracted from a variety of essential oils of flowers and plants. It has the advantages of easy availability and small side effects, and has received attention in many industries in recent years.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a new pharmaceutical application of nerolidol.

In order to solve the above technical problems, embodiments of the present invention provide the application of nerolidol in the preparation of a medicament for treating fungal keratitis.

In the above pharmaceutical uses provided by the present invention, nerolidol is used to inhibit the growth of corneal fungi, thereby achieving the therapeutic effect of inhibiting the degree of corneal inflammation. Wherein, the corneal fungus is Aspergillus fumigatus.

In the above pharmaceutical use provided by the present invention, the nerolidol inhibits the LOX-1/IL-1β signaling pathway in corneal tissue. More specifically, the LOX-1/IL-1β signaling pathway in the corneal tissue is the LOX-1/IL-1β signaling pathway in human corneal epithelial cells stimulated by Aspergillus fumigatus.

Embodiments of the present invention also provide a medicament for treating fungal keratitis, the medicament comprising nerolidol and pharmaceutically acceptable excipients.

The present invention proposes the application in the preparation of a medicament for treating fungal keratitis for the first time. In the present invention, different concentrations of nerolidol and Aspergillus fumigatus spores are co-cultured for 24 hours to study the inhibitory effect of different concentrations of nerolidol on Aspergillus fumigatus. In the in vivo study, a fungal keratitis model was established in C57BL/6 mice, and the effects of nerolidol on the corneal clinical score, neutrophil recruitment, and LOX-1/IL-1β signaling pathway in the model mice were studied. In an in vitro study, the inhibition of LOX-1/IL-1β signaling pathway by nerolidol was validated using a human corneal epithelial cell model stimulated by Aspergillus fumigatus. The above experimental results confirm that nerolidol can inhibit the growth of Aspergillus fumigatus in the cornea, and has a therapeutic effect on fungal keratitis both in vivo and in vitro.

Description of drawings

Fig. 1 is the result graph of the optical density value of Aspergillus fumigatus spores cultivated in different concentrations of nerolidol for 24 hours in Example 1;

Fig. 2 is the result diagram of fungal cell wall staining in the treatment group of different concentrations of nerolidol in Example 1;

Figure 3 is a comparison diagram of the mouse cornea of the PBS-treated infection group and the nerolidol-treated infection group under a slit lamp microscope 3 days after the establishment of the fungal keratitis mouse model in Example 2;

4 is a graph showing the results of clinical scoring of the PBS-treated infection group and the nerolidol-treated infection group 3 days after the establishment of the fungal keratitis mouse model in Example 2;

Fig. 5 is the fluorescence graph that the PBS-treated infection group and the nerolidol-treated infection group adopt immunofluorescence technique to detect inflammatory cell recruitment 2 days after the establishment of the fungal keratitis mouse model in Example 2;

Fig. 6 is the myeloperoxidase activity score result of PBS-treated infection group and nerolidol-treated infection group 2 days after the establishment of the fungal keratitis mouse model in Example 2;

Fig. 7 is the result diagram of adopting polymerase chain reaction to detect the effect of nerolidol on the expression of LOX-1 mRNA in the cornea of fungal keratitis model mice in Example 2;

Fig. 8 is the result diagram of adopting Western blot to detect the effect of nerolidol on the secretion of LOX-1 protein in the cornea of fungal keratitis model mice in Example 2;

Fig. 9 is the result diagram that adopts polymerase chain reaction to detect the effect of nerolidol on the expression of IL-1β mRNA in the cornea of fungal keratitis model mice in Example 2;

Figure 10 is a result diagram of the effect of nerolidol on the secretion of IL-1β protein in the cornea of fungal keratitis model mice using Western blot in Example 2;

Figure 11 is a result diagram of the effect of nerolidol on the expression of LOX-1 mRNA in human corneal epithelial cells stimulated by fungi using polymerase chain reaction in Example 3;

Fig. 12 is the result diagram of using western blot to detect the effect of nerolidol on the secretion of LOX-1 protein in human corneal epithelial cells stimulated by fungi in Example 3;

Figure 13 is a result diagram of the effect of nerolidol on the expression of IL-1β mRNA in human corneal epithelial cells stimulated by fungi using polymerase chain reaction in Example 3;

14 is a graph showing the results of using Western blotting in Example 3 to detect the effect of nerolidol on the secretion of IL-1β protein in fungal-stimulated human corneal epithelial cells.

Detailed ways

In order to make the objectives, technical solutions and advantages of the present invention clearer, each embodiment of the present invention will be described in detail below. However, those of ordinary skill in the art can appreciate that, in the various embodiments of the present invention, many technical details are set forth in order for the reader to better understand the present application. However, even without these technical details and various changes and modifications based on the following embodiments, the technical solutions claimed in the claims of the present application can be realized.

Nerolidol is a natural hemisesterpene alcohol in a variety of essential oils of flowers and plants, and has certain medicinal potential. The specific embodiments of the present invention mainly discuss: the inhibitory effect of nerolidol on the growth of Aspergillus fumigatus, and the therapeutic effect on fungal keratitis in vivo and in vitro. In the in vivo study, a fungal keratitis model was established in C57BL/6 mice, and the effect of nerolidol on the corneal clinical score, neutrophil recruitment, and LOX-1/IL-1β signaling pathway of the model mice was studied. In an in vitro study, the inhibition of LOX-1/IL-1β signaling pathway by nerolidol was validated using a human corneal epithelial cell model stimulated by Aspergillus fumigatus.

The experimental scheme and results made by the embodiment of the present invention are as follows:

The conidia were dissolved in Sabouraud medium to prepare a mixed solution with a concentration of 5×10 ³ cfu/ml, and cultured with different concentrations of nerolidol in a 37°C incubator for 24 hours. Optical density at 460 nm was measured. Subsequently, the hyphal cell walls were fluorescently stained. The results showed that compared with the control group, nerolidol at a concentration of 100 μM could significantly inhibit the growth of Aspergillus fumigatus, and at 800 μM, it could completely inhibit the spore germination of Aspergillus fumigatus.

In the in vivo experiment, the fungal keratitis mouse model was established by intrastromal injection, and different groups of model mice were intervened by subconjunctival injection of nerolidol or the same amount of PBS. taking photos. The mouse eyeballs and corneas were collected on the second day, and the recruitment of corneal neutrophils was detected by immunofluorescence and myeloperoxidase (MPO) assays; on the third day, the mouse corneas were collected and analyzed by polymerase chain reaction In the experiment, the mRNA expressions of LOX-1 and IL-1β in the cornea were detected, and the protein levels of LOX-1 and IL-1β in the cornea were detected by western blotting. The results showed that, compared with the PBS control group, the nerolidol-intervention fungal infection group reduced the degree of corneal inflammation, decreased neutrophil recruitment, and decreased LOX-1/IL-1β mRNA and protein levels.

In vitro experiments, human corneal epithelial cells were used to validate the anti-inflammatory mechanism studied in vivo. Human corneal epithelial cells were stimulated by Aspergillus fumigatus conidia, followed by intervention in different groups with nerolidol or PBS, and cells were harvested at 8 or 16 hours. Cells were collected at 8 hours for detection of LOX-1 and IL-1β mRNA expression in human corneal epithelial cells by polymerase chain reaction assay. After 16 hours, cells were harvested for detection of LOX-1 and IL-1β protein levels in human corneal epithelial cells by western blotting. The results were consistent with the results of the mouse keratitis model. Compared with the PBS control, nerolidol intervention could inhibit LOX-1/IL-1β in the Aspergillus fumigatus stimulated group.

Embodiment 1 Nerolidol inhibits the growth of Aspergillus fumigatus

1. Experimental materials

1.1 Experimental drugs: nerolidol (purchased from selleck company); Calcoflor fluorescent whitening agent (purchased from Sigma-aldrich company)

1.2 Experimental fungi: 3.0772 strains of Aspergillus fumigatus (China General Microorganism Culture Collection and Management Center)

2. Experimental method

2.1 Fungal Preparation

The Aspergillus fumigatus strain was inoculated on Sabouraud's medium, and cultured in a constant temperature incubator at 37°C for 2-3 days. The hyphae and conidia were scraped and put into 5ml sterile PBS to form a mixed solution, and the hyphae were removed by filtration with sterile gauze. Centrifuge at 4500 rpm at 4°C for 10 minutes, discard the supernatant, add 5 ml of bacterial PBS to the pellet to resuspend to form a conidia suspension. The concentration of conidia was adjusted to 5 x 106 ^cfu /ml with sterile PBS.

2.2 Minimum inhibitory concentration experiment

The conidia suspension was diluted to 5 x ¹⁰³ cfu/ml with Sabouraud's broth. The nerolidol was dissolved in Sabouraud culture medium containing conidia, and diluted twice, so that the concentration of nerolidol was 800 μM at the maximum and 50 μM at the minimum. The volume of 100 μl per well was distributed into a 96-well plate, and the number of repetitions was 3. After culturing in a constant temperature incubator at 37°C for 24 hours, the optical density value at 460 nm was measured with a microplate reader. Then, centrifuge at 4°C and 4500rpm for 5 minutes, discard the supernatant, add Calcoflor fluorescent whitening agent to each well to fluorescently stain the hyphal cell wall, and observe the hyphae of each well with a fluorescence microscope after 30 minutes, and capture digital image.

3. Experimental results

The minimum inhibitory concentration method was used to detect nerolidol inhibiting the growth of Aspergillus fumigatus. Fig. 1 is the result graph of the optical density value of Aspergillus fumigatus spores cultivated in different concentrations of nerolidol for 24 hours; Fig. 2 is the result graph of fungal cell wall staining in the treatment groups of different concentrations of nerolidol (the numbers in the lower right corner of the figure) represents the concentration of nerolidol, in μM).

As shown in Figure 1, compared with the control group, nerolidol significantly inhibited the growth of Aspergillus fumigatus at 100 μM (P<0.01). As shown in Figure 2, the blue fluorescence represents the hyphal cell wall of Aspergillus fumigatus, and the results show that the concentration that completely inhibits the germination of Aspergillus fumigatus spores is 800 μM.

Example 2 Effects of nerolidol on corneal clinical score, neutrophil recruitment and LOX-1/IL-1β signaling pathway in fungal keratitis model mice.

1. Experimental materials

1.1 Experimental drug: nerolidol (purchased from selleck company)

1.2 Experimental animals: C57BL/6 mice (purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd.)

1.3 Experimental fungi: 3.0772 strains of Aspergillus fumigatus (China General Microorganism Culture Collection and Management Center)

2. Experimental method

2.1 Model mice

8-week-old healthy clean-grade C57BL/6 female mice were selected as the research subjects. Eye diseases were excluded by slit lamp examination before the experiment, and the right eye was selected as the experimental eye. All manipulations of experimental mice were in compliance with the Chinese Ministry of Science and Technology's guidelines for humanized treatment of experimental animals (vGKFCZ-2006–398), and in line with the Association for Research in Vision and Ophthalmology (ARVO) Principles and Standards for the Use of Animals in Ophthalmology and Vision Research.

2.2 Effects of nerolidol on corneal clinical score, neutrophil recruitment and LOX-1/IL-1β signaling pathway in fungal keratitis model mice

C57BL/6 mice were randomly divided into control group, PBS-treated fungal infection group, nerolidol-treated group and nerolidol-treated fungal infection group. After the mice were anesthetized by intraperitoneal injection of 8% chloral hydrate, 2.5 μl of a conidia suspension with a concentration of 2.5×10 ⁶ cfu/ml was injected into the fungal infection group and the nerolidol treated fungal infection group using a microsyringe in mouse corneal stroma. After 30 minutes, 5 μL of PBS was injected into the subconjunctiva of the mice in the control group and the fungal infection group, and 5 μL of nerolidol dissolved in PBS was injected into the subconjunctiva of the mice in the nerolidol-treated group and the nerolidol-treated fungal infection group. (200 μM). After modeling, the mouse corneas were observed and photographed with a slit lamp microscope every day. One day and three days after modeling, the mice in each group were injected with PBS or nerolidol under the conjunctiva for intervention. Mice were sacrificed on the 2nd day after modeling, eyeballs were taken for immunofluorescence experiments, and corneas were taken for myeloperoxidase detection. The mice were sacrificed on the 3rd day after modeling, and the corneas were used for polymerase chain reaction and western blotting experiments to detect the expressions of LOX-1 and IL-1β in mouse corneas.

3. Experimental results

3.1 The effect of nerolidol on the corneal clinical score of fungal keratitis model mice

Figure 3 is a comparison of the cornea of the PBS-treated infection group and the nerolidol-treated infection group under slit lamp microscope 3 days after the establishment of the fungal keratitis mouse model; Figure 4 is the fungal keratitis mouse model 3 days after establishment, the results of the clinical score of the PBS-treated infection group and the nerolidol-treated infection group.

As shown in Figures 3 and 4, compared with the PBS-treated infected group, the inflammation degree and clinical score of the mouse corneas in the nerolidol-treated infected group were significantly reduced.

3.2 The effect of nerolidol on the recruitment of inflammatory cells in the cornea of fungal keratitis model mice

Figure 5 shows the fluorescence images of the PBS-treated infection group and the nerolidol-treated infection group using immunofluorescence technique to detect the recruitment of inflammatory cells 2 days after the establishment of the fungal keratitis mouse model; Figure 6 is the fungal keratitis mice 2 days after the model was established, myeloperoxidase activity scores in the PBS-treated infection group and the nerolidol-treated infection group;

As shown in Figures 5 and 6, compared with the PBS-treated infection group, nerolidol treatment can significantly reduce the number of neutrophils recruited and myeloperoxidase in the cornea of fungal keratitis model mice infected for 2 days activity.

3.3 The effect of nerolidol treatment on the secretion of corneal LOX-1/IL-1β in fungal keratitis model mice

Fig. 7 is a graph showing the results of using polymerase chain reaction to detect the effect of nerolidol on the expression of LOX-1 mRNA in the cornea of fungal keratitis model mice; Figure 9 is the result of the effect of nerolidol on the expression of IL-1β mRNA in the cornea of fungal keratitis model mice by polymerase chain reaction. ; Figure 10 is the result of detecting the effect of nerolidol on the secretion of IL-1β protein in the cornea of fungal keratitis model mice by western blotting.

As shown in Figure 7 and Figure 8, polymerase chain reaction experiments showed that compared with the PBS-treated infection group, the nerolidol-treated fungal infection group had significantly higher mRNA expression of LOX-1 and IL-1β in the cornea of the mice reduce. As shown in Figure 9 and Figure 10, Western blotting experiments showed that the protein levels of LOX-1 and IL-1β in the cornea of the mice in the fungal infection group treated with nerolidol were significantly lower than those in the infection group treated with PBS.

Example 3 Nerolidol can block the LOX-1/IL-1β pathway in fungal-stimulated human corneal epithelial cells

1. Experimental materials

1.1 Experimental drug: nerolidol (purchased from selleck company)

1.2 Experimental cells: human corneal epithelial cells (purchased from the Ocular Surface Laboratory of Zhongshan Ophthalmic Center)

1.3 Experimental fungi: 3.0772 strains of Aspergillus fumigatus (China General Microorganism Culture Collection and Management Center)

2. Experimental method

2.1 Cell model experiment

Human corneal epithelial cells were placed in a 37°C, 5% _CO2 incubator and cultured until the cell density reached 80%. The cells were divided into control group, PBS-treated fungal stimulation group, nerolidol-treated group and nerolidol-treated fungal stimulation group. Nerolidol (final concentration 200 μM) was added to the cell culture medium of the nerolidol-treated group and the fungal stimulation group treated with nerolidol, and the same amount of PBS was added to the cell culture medium of the control group and the fungal stimulation group. Subsequently, Aspergillus fumigatus conidia were added to the cell cultures of the PBS-treated fungal-stimulated group and the nerolidol-treated fungal-stimulated group. After 8 hours of incubation in a 37°C incubator, the cells were collected for polymerase chain reaction experiments to detect the mRNA expression of LOX-1 and IL-1β. After 16 hours of fungal stimulation, cells were harvested for western blotting to detect the protein levels of LOX-1 and IL-1β.

3. Experimental results

Figure 11 is the result of using polymerase chain reaction to detect the effect of nerolidol on the expression of LOX-1 mRNA in human corneal epithelial cells stimulated by fungi; The result graph of the effect of LOX-1 protein secretion in corneal epithelial cells; Fig. 13 is the result graph of the effect of nerolidol on the expression of IL-1β mRNA in fungal-stimulated human corneal epithelial cells by polymerase chain reaction; Fig. 14 is the result of detecting the effect of nerolidol on the secretion of IL-1β protein in human corneal epithelial cells stimulated by fungi by western blot;

As shown in Figure 11 and Figure 12, polymerase chain reaction experiments showed that compared with the fungal stimulation group treated with PBS, the mRNA of LOX-1 and IL-1β in the human corneal epithelial cells of the fungal stimulation group treated with nerolidol expression was significantly reduced. As shown in Figure 13 and Figure 14, Western blotting experiments showed that the protein levels of LOX-1 and IL-1β in human corneal epithelial cells in the fungal stimulation group treated with nerolidol were significantly lower than those in the fungal stimulation group treated with PBS .

Those skilled in the art can understand that the above-mentioned embodiments are specific examples for realizing the present invention, and in practical applications, various changes in form and details can be made without departing from the spirit and the spirit of the present invention. scope.

Claims (6)

1. Application of nerolidol in the preparation of a medicament for treating fungal keratitis. 2. application according to claim 1 is characterized in that, described nerolidol inhibits corneal fungal growth. 3. application according to claim 2, is characterized in that, described corneal fungus is Aspergillus fumigatus. The application according to claim 2, wherein the nerolidol inhibits the LOX-1/IL-1β signaling pathway in corneal tissue. The application according to claim 3, wherein the LOX-1/IL-1β signaling pathway in the corneal tissue is the LOX-1/IL-1β signaling pathway in human corneal epithelial cells stimulated by Aspergillus fumigatus. 6. A medicine for treating fungal keratitis, characterized in that, comprising nerolidol and pharmaceutically acceptable adjuvants.

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