Application of soy isoflavones in preparing medicine for treating keratoconus

Abstract

The present invention provides the use of soy isoflavones in the treatment of keratoconus. The present invention finds that soy isoflavones are non-toxic and do not inhibit cell growth in corneal stromal cells. Furthermore, soy isoflavones are easy to prepare and store, have wide clinical applications, and exhibit minimal side effects, making them suitable for formulation into eye drops. Experimental results demonstrate that soy isoflavones effectively inhibit the release of matrix metalloproteinase 1 and matrix metalloproteinase 3 from corneal stromal cells. Furthermore, soy isoflavones effectively enhance the elastic modulus of corneal stromal tissue.

Description

Technical Field

[0001] The present invention belongs to the technical field of preparation and application of biological medicines, and particularly relates to the application of soy isoflavones in the preparation of medicines for treating keratoconus. Background Art

[0002] Keratoconus is a bilateral, non-inflammatory corneal disease characterized by corneal dilation and thinning, which can lead to irregular astigmatism and myopia. In severe cases, it can cause vision loss or even blindness. The dilation and thinning of the corneal stroma in keratoconus are associated with excessive release of matrix metalloproteinases (primarily 1 and 3) in the corneal stroma, but the underlying pathogenesis remains unclear. Numerous reports suggest that environmental changes caused by upregulation of inflammatory mediators such as interleukin-6 (IL-6) may be associated with the release of corneal matrix metalloproteinases.

[0003] Although corneal cross-linking surgery can be performed in the early stages to slow progression, some patients still require corneal transplantation in the later stages, and the treatment effect is still far from what patients need. Currently, there is no drug that can clearly prevent or control the progression of the disease. Therefore, the development of a drug to treat keratoconus by inhibiting the release of matrix metalloproteinases in a hyperinflammatory environment provides a possibility for medical treatment of keratoconus and is of great significance in enriching the treatment options for keratoconus. Summary of the Invention

[0004] The purpose of the present invention is to provide the use of soy isoflavones in treating keratoconus, so as to fill the gap in the medical treatment of keratoconus.

[0005] The present invention first provides a new application of soy isoflavones, which is application in treating keratoconus.

[0006] The present invention also provides another use of soybean isoflavones, which is use in preparing medicine for treating keratoconus.

[0007] A drug for treating keratoconus, comprising soy isoflavones at a pharmacologically effective concentration;

[0008] The medicine is a liquid medicine, such as eye drops.

[0009] The present invention discovered that soy isoflavones (SI) are non-toxic when applied to corneal stromal cells, and cell growth is not inhibited. Furthermore, soy isoflavones are easy to prepare and store, have a wide range of clinical applications, and have minimal side effects, making them suitable for use as eye drops. Experimental results showed that soy isoflavones effectively inhibited the release of matrix metalloproteinase 1 (MMP1) and matrix metalloproteinase 3 (MMP3) from corneal stromal cells. Furthermore, soy isoflavones effectively enhanced the elastic modulus of corneal stromal tissue (i.e., they have the effect of increasing tissue hardness). BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 :Comparison of cell morphology;

[0011] Figure 2 :Soy isoflavones inhibit the gene expression of matrix metalloproteinases 1 and 3,

[0012] Figure 3 :ELISA test of soy isoflavones inhibiting the extracellular release of matrix metalloproteinases 1 and 3 in corneal stromal cells;

[0013] Figure 4 :WB image of soy isoflavones inhibiting the intracellular expression of matrix metalloproteinases 1 and 3;

[0014] Figure 5 : Detection diagram of MMP1 and MMP3 expression levels.

[0015] Figure 6 : Soy isoflavones improve the elastic modulus of corneal stromal tissue.

[0016] Figure 7 :Soy isoflavones have a combined effect of antagonizing collagenase and inflammation in mice. DETAILED DESCRIPTION

[0017] Soy isoflavones, used in this invention, are natural phytoestrogens primarily found in soybeans and their products. They exhibit certain estrogen-like activity in the human body and have a wide range of impacts on human health. In terms of women's health, soy isoflavones may help alleviate menopausal symptoms such as hot flashes, sweating, and insomnia. They may also benefit women's skin and bone health.

[0018] The present invention is described in detail below with reference to the embodiments and accompanying drawings.

[0019] Example 1: Soy isoflavones have no effect on cell morphology and cell activity

[0020] Primary human corneal stromal cells were cultured in 6-cm culture dishes using a medium supplemented with 10% fetal bovine serum (DF12 + 10% fetal bovine serum). The medium was changed daily and passaged every 5 days. At the third passage, cells were treated with either no treatment, IL6 50 ng / ml, or IL6 50 ng / ml + soy isoflavones SI 5 µM. Cell morphology was observed and the supernatant was collected for CCK8 cytotoxicity assay. The results are shown in Figure 2. Figure 1 and Figure 2 As shown, the results showed that there was no significant change in the morphology of human corneal stromal cells after treatment with soy isoflavones SI, and there was no growth inhibition.

[0021] Example 2: Soy isoflavones inhibit the release of matrix metalloproteinase 1 and matrix metalloproteinase 3 at the gene level

[0022] Primary human corneal stromal cells were cultured in 6-cm culture dishes using DF12 medium supplemented with 10% fetal bovine serum. The medium was changed daily, and passages were performed every 5 days. At the third passage, cells were treated with either no treatment, IL6 50 ng / ml, or IL6 50 ng / ml plus SI 5 µm. After harvesting the cells using Trizol reagent, quantitative PCR was performed to detect the expression of MMP1 (Table 1) and MMP3 (Table 2). Inflammation slightly increased the expression of corneal matrix metalloproteinases, while SI significantly inhibited the expression of MMP1 and MMP3 genes ( Figure 2 ).

[0023] Table 1: Data table of the effects of soy isoflavones on the expression of MMP1

[0024]

[0025] Table 2: Effects of soy isoflavones on MMP3 expression

[0026]

[0027] The cell culture supernatant was collected and the content of matrix metalloproteinases 1 and 3 released into the extracellular space was detected using an Elisa kit. It was found that under the action of inflammatory mediators, the content of MMP1 and MMP3 released from the supernatant of corneal stromal cells increased. However, if soy isoflavones were added on the basis of inflammation, the release of MMP1 and MMP3 would be inhibited (Table 3, Table 4). This shows that soy isoflavones have an inhibitory effect on the release of extracellular MMP1 and MMP3 ( Figure 3 ) .

[0028] Table 3: Effects of soy isoflavones on extracellular MMP1 expression

[0029]

[0030] Table 4: Effects of soy isoflavones on extracellular MMP3 expression

[0031]

[0032] In addition, WB electrophoresis and fluorescence quantitative staining were performed on the cultured cells to detect the expression of MMP1 and MMP3, and it was found that SI had the effect of inhibiting their protein and gene expression ( Figure 5 ).

[0033] Example 3: Effects of soy isoflavones on corneal stroma

[0034] Corneal stromal tissue was collected and cultured in vitro after femtosecond surgery. The tissues were treated with either no treatment, IL6 50ng / ml, or IL6 50ng / ml + SI 5µm. The elastic modulus was measured using a biaxial biomechanical measuring instrument. It was observed that under the action of inflammatory mediators, the elastic modulus of the corneal stromal lens decreased. However, when SI was added in addition to inflammatory mediators, the elastic modulus of the corneal stromal lens increased, even exceeding the control level. This suggests that SI significantly enhances the elastic modulus of corneal stromal tissue, strengthening its hardness ( Figure 6 ).

[0035] An animal model of corneal thinning was established using a C57 mouse model. The mice were divided into a control group, a collagenase combined with inflammatory mediator treatment group, and a collagenase combined with inflammatory mediator treatment followed by drug treatment group. From day 1 to day 7 after treatment, the mice in the drug treatment group were continuously treated with drugs. Seven days later, changes in corneal thickness were measured using optical coherence tomography (OCT). The collagenase combined with inflammatory mediator treatment group showed statistically significant corneal thinning compared to the control group, while the combined treatment group showed no statistically significant difference in corneal thickness compared to the control group. Figure 7 ).

[0036] The results showed that soy isoflavones effectively changed the elastic modulus of corneal stromal tissue and enhanced the hardness of corneal stromal lens; in addition, it could antagonize collagenase and inflammatory mediators in mouse models, and had the effect of delaying corneal thinning under the action of anti-collagenase and inflammatory mediators.

Claims

1. Use of soy isoflavones as the sole active ingredient in the preparation of a medicament for treating keratoconus, characterized in that: The drug can inhibit the excessive release of matrix metalloproteinases in the corneal stroma and enhance the elastic modulus of the corneal stroma tissue.

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