Application of silybin in the preparation of drugs for preventing and / or treating proliferative vitreoretinopathy

Abstract

The present invention relates to the use of silibinin in the preparation of a drug for preventing and / or treating proliferative vitreoretinopathy, and belongs to the field of biomedicine technology. The present invention verifies through cell level and animal level that silibinin can prevent the epithelial-mesenchymal transformation process of retinal pigment epithelial cells, inhibit the formation of proliferative vitreoretinopathy proliferative membrane, thereby preventing the progression of proliferative vitreoretinopathy, and providing a new, effective and non-toxic drug for the treatment of clinical proliferative vitreoretinopathy.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of silybin in preparing a drug for preventing and / or treating proliferative vitreoretinopathy. Background Art

[0002] Proliferative vitreoretinopathy (PVR) is a refractory blinding eye disease, a serious complication of rhegmatogenous retinal detachment, and the most common cause of failure in retinal detachment reattachment surgery. About 75% of PVR patients will experience retinal reattachment after the initial retinal detachment repair surgery. At present, surgery is the main treatment for clinical PVR, and the method is surgical removal of the proliferative membrane. It is reported that only 40% to 80% of patients achieve retinal function reattachment after PVR surgery, and there is a certain recurrence rate after surgery. The reason is that the timing of surgical intervention is often after the formation of a dense PVR proliferative membrane, which is in the late stage of the PVR pathological process. In addition, surgery cannot prevent the continuous proliferation and migration of retinal pigment epithelial (RPE) cells and cannot completely block the pathological process of PVR, which leads to the continuous and repeated generation of PVR proliferative membrane.

[0003] RPE cells are the most common cell component in the proliferative membrane of PVR, existing in almost all epiretinal membranes. At the same time, RPE cells are also the main cell type involved in the formation of proliferative membrane tension. The epithelial-mesenchymal transition (EMT) of RPE cells is the core pathological mechanism of PVR formation. During the EMT process, RPE cells migrate to the surface of the retina, and the cells transform from polygonal epithelial cell morphology to long spindle-shaped mesenchymal cell morphology, while expressing extracellular matrix-related genes and mesenchymal-related genes, such as COL1A1, Fibronectin1 and MMP2; finally, they transform into α-smooth muscle actin (αSMA)-positive myofibroblasts, which are the main cell type mediating epiretinal membrane contraction.

[0004] Studies have shown that existing anti-inflammatory and anti-proliferative drugs fail to treat the core pathological mechanism of PVR and have not been used and shown to be effective in clinical practice. Therefore, there is an urgent need for preventive or early anti-PVR drugs that can target RPE cell EMT.

[0005] Silibinin, chemical name: 2,3-dihydro-3-(4-hydroxy-3-methoxyphenyl)-2-hydroxymethyl-6-(3,5,7-trihydroxy-4-oxobenzopyran-2-yl)benzodioxane, is a flavonoid glycoside compound extracted from the seeds of the Asteraceae plant, Silybum marianum. Existing literature has reported that the main pharmacological effects of silibinin include anti-fibrosis, anti-tumor, anti-inflammatory, and antioxidant effects. It can be used in the treatment of diseases such as pulmonary fibrosis, renal fibrosis, breast cancer, prostate cancer, and abrin-induced hepatotoxicity. So far, there has been no report on the anti-proliferative vitreoretinopathy effect of silibinin. It is impossible to infer whether silibinin has an anti-proliferative vitreoretinopathy effect based on the known properties of silibinin. Summary of the invention

[0006] The purpose of the present invention is to overcome the shortcomings of the prior art and provide the use of silybin in the preparation of a drug for preventing and / or treating proliferative vitreoretinopathy.

[0007] To achieve the above object, the technical solution adopted by the present invention is:

[0008] In a first aspect, the present invention provides the use of silybin in the preparation of a drug for preventing and / or treating proliferative vitreoretinopathy.

[0009] The present invention is the first to discover that silybin can effectively prevent the progression of proliferative vitreoretinopathy (PVR) at the animal experimental level. By treating the PVR animal model with silybin, it is found that silybin can prevent retinal shrinkage and detachment and inhibit the formation of proliferative membranes, thereby preventing proliferative vitreoretinopathy.

[0010] As a preferred embodiment of the application of the present invention, the proliferative vitreoretinopathy is caused by rhegmatogenous retinal detachment.

[0011] As a preferred embodiment of the application of the present invention, the silybin includes at least one of silybin A and silybin B.

[0012] As a preferred embodiment of the application of the present invention, the silybin includes silybin A and silybin B.

[0013] As a preferred embodiment of the application of the present invention, the mass ratio of silybin A to silybin B in the silybin is silybin A:silybin B=1:1.

[0014] As a preferred embodiment of the application of the present invention, the dosage form of the drug for preventing and / or treating proliferative vitreoretinopathy is at least one of capsules, tablets, oral preparations, microcapsule preparations, injections, suppositories, sprays, ointments, gels, solutions, powders, lotions, tinctures, oils, creams and aerosols.

[0015] As a preferred embodiment of the application of the present invention, the drug for preventing and / or treating proliferative vitreoretinopathy is used for vitreous cavity injection.

[0016] As a preferred embodiment of the application of the present invention, the dosage of the drug for preventing and / or treating proliferative vitreoretinopathy injected into the vitreous cavity is 100-200 μM silybin. The present invention found in the experimental process that 100-200 μM silybin can significantly inhibit the expression of mesenchymal markers of RPE cells, and the higher the concentration of silybin, the more significant the inhibitory effect. When the dosage of silybin injected into the vitreous cavity for treating proliferative vitreoretinopathy is 100-200 μM, the progression of PVR can be effectively prevented.

[0017] In a second aspect, the present invention provides the use of silybin in the preparation of a drug for organizing epithelial-mesenchymal transition of retinal pigment epithelial cells.

[0018] The present invention firstly finds that silybin can effectively prevent the epithelial-mesenchymal transition (EMT) of retinal pigment epithelial cells (retinal pigment epithelium, RPE) at the cellular level. By treating RPE with silybin, it is found that silybin can prevent the transformation of RPE cell morphology into mesenchymal cells and inhibit the expression of mesenchymal markers, thereby inhibiting the EMT process of RPE.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention verifies through cell level and animal level that silybin can inhibit the expression of mesenchymal markers of retinal pigment epithelial cells, prevent the epithelial-mesenchymal transformation process of retinal pigment epithelial cells, inhibit the formation of proliferative vitreoretinopathy proliferative membrane, alleviate the phenomenon of retinal shrinkage and tractional retinal detachment caused by PVR, thereby achieving the effect of preventing proliferative vitreoretinopathy, and providing a new, effective and non-toxic drug for the treatment of clinical proliferative vitreoretinopathy. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The microscope images of human retinal pigment epithelial cells in each experimental group in Experimental Example 1;

[0022] Figure 2 The results of the detection of the mRNA expression levels of mesenchymal markers in human retinal pigment epithelial cells in each experimental group in Experimental Example 1, wherein A is the quantitative result of the mRNA expression level of the mesenchymal marker COL1A1, B is the quantitative result of the mRNA expression level of the mesenchymal marker MMP2, and C is the quantitative result of the mRNA expression level of the mesenchymal marker Fibronectin1;

[0023] Figure 3 The results of the detection of the protein expression levels of mesenchymal markers in human retinal pigment epithelial cells in each experimental group in Experimental Example 1;

[0024] Figure 4 The quantitative analysis results of the mesenchymal marker protein expression levels of human retinal pigment epithelial cells in each experimental group in Experimental Example 1, wherein A is the quantitative result of the mesenchymal marker COL1A1 protein expression level, B is the quantitative result of the mesenchymal marker MMP2 protein expression level, C is the quantitative result of the mesenchymal marker Fibronectin1 protein expression level, and D is the quantitative result of the mesenchymal marker N-cadherin protein expression level;

[0025] Figure 5 The results of fluorescent immunostaining of mesenchymal markers of human retinal pigment epithelial cells in each experimental group in Experimental Example 1, wherein A is incubated with Fibronectin1 primary antibody, and B is incubated with αSMA primary antibody, and the scale bar in the figure is 100 μm;

[0026] Figure 6 Fundus photographs and PVR rating results of rats in each experimental group in Experimental Example 2, where A is fundus photographs of rats at different times after injection, B is the PVR rating result on the 7th day after injection, C is the PVR rating result on the 14th day after injection, and D is the PVR rating result on the 21st day after injection;

[0027] Figure 7 The results of HE staining and immunofluorescence staining of the eyeballs of rats in each experimental group in Experimental Example 2, where A is the HE staining result of the rat eyeballs, and B is the immunofluorescence staining result of the eyeballs;

[0028] In the above figures, “ns” means P>0.05, “*” means P<0.05, “**” means P<0.01, “***” means P<0.001, and “****” means P<0.0001. DETAILED DESCRIPTION

[0029] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0030] Unless otherwise specified, the reagents and consumables used in the following experimental examples can be obtained through commercial channels.

[0031] Human retinal pigment epithelial cells ARPE-19 ( ATCC No.CRL-2302 provided by American Type Culture Collection (ATCC).

[0032] The serum-containing DMEM / F-12 medium is DMEM / F-12 medium containing 10 v / v% fetal bovine serum, 100 μg / ml penicillin solution, and 100 μg / ml streptomycin solution.

[0033] Silybin was provided by Sigma, catalog number PHL89280.

[0034] Experimental Example 1 In vitro anti-EMT experiment of silybin

[0035] In order to verify the effect of silybin on human retinal pigment epithelial cells, the anti-epithelial-mesenchymal transition (EMT) effect of silybin in vitro was evaluated by cell experiments. The specific scheme is as follows:

[0036] (1) Experimental materials and treatment

[0037] ARPE-19 cells were selected as the experimental subjects and cultured at 37°C and 5% CO 2 , cultured in DMEM / F-12 medium containing serum.

[0038] In order to simulate the EMT process of ARPE-19 cells, 10 ng / ml transforming growth factor-β1 (TGFβ1) was added during cell culture to induce EMT in ARPE-19. A total of 6 experimental groups were set up in this experiment, including a control group and a treatment group, as shown in Table 1. ARPE-19 cells were cultured until adherent, pretreated with silybin for 1 hour, and then treated with TGFβ1. After 48 hours of culture, various indicators were detected.

[0039] Table 1 Treatment of each experimental group

[0040] Group TFGβ1 treatment Silybin treatment Control group 1 0 0μM Control group 2 10ng / ml 0μM Treatment Group 1 10ng / ml 50μM Treatment Group 2 10ng / ml 100μM Treatment Group 3 10ng / ml 150μM Treatment Group 4 10ng / ml 200μM

[0041] (2) Observe the morphology of cells in each experimental group

[0042] The morphology of cells in each experimental group was observed by optical microscopy. Figure 1 .

[0043] like Figure 1As shown, the ARPE-19 cells in the control group 2 transformed from polygonal to long spindle-shaped, indicating that TGFβ1 can induce ARPE-19 to transform from epithelial cells to mesenchymal cells; while the ARPE-19 cells treated with silybin tended to maintain a polygonal shape, and as the concentration of silybin increased, the polygonal shape of the cells became clearer, indicating that silybin can effectively prevent RPE cells from transforming into mesenchymal cells.

[0044] (3) Detection of mesenchymal marker mRNA expression in cells of each experimental group

[0045] Real-time fluorescence quantitative PCR was used to detect the mRNA expression levels of mesenchymal markers in each experimental group. The mesenchymal markers included COL1A1, Fibronectin1, and MMP2, and the internal reference was GAPDH.

[0046] The RNA of cells in each experimental group was extracted by Trizol method, and the RNA was reverse transcribed into cDNA. The mRNA expression levels of mesenchymal markers COL1A1, Fibronectin1, and MMP2 were detected by real-time fluorescence quantitative PCR, with GAPDH as the internal reference. The results are shown in Figure 2 .

[0047] like Figure 2 As shown, compared with the control group 2, the mRNA expression levels of mesenchymal markers COL1A1, MMP2 and Fibronectin1 in the ARPE-19 cells in the treatment group were decreased, indicating that silybin can significantly downregulate the transcription of mesenchymal markers in RPE cells, and as the concentration of silybin increases, the transcription levels of mesenchymal markers COL1A1 and MMP2 are lower.

[0048] (4) Detection of mesenchymal marker protein expression in cells of each experimental group

[0049] The protein expression levels of mesenchymal markers in each experimental group were detected by Western blot (WB). The mesenchymal markers included COL1A1, Fibronectin1, MMP2, and N-cadherin, and the internal reference was GAPDH.

[0050] The total protein of each experimental group was extracted by physical extraction, and the protein concentration was determined by BCA method and the same concentration was obtained by dilution method. SDS-PAGE electrophoresis, membrane transfer, incubation with primary antibody, blocking, incubation with secondary antibody, color development, WB detection, and grayscale analysis of WB imaging results were performed by Image J. The primary antibodies used included MMP2 (1:1000), COL1A1 (1:1000), FN (1:1000), N-cadherin (1:10000) and GAPDH (1:20000). The results of WB detection and analysis are shown in Figure 3 .

[0051] like Figure 3 As shown, compared with the control group 2, the protein expression levels of mesenchymal markers COL1A1, MMP2, Fibronectin1 and N-cadherin in the ARPE-19 cells in the treatment group were decreased, indicating that silybin can significantly downregulate the protein expression of mesenchymal markers, and as the concentration of silybin increases, the protein expression level of mesenchymal markers decreases.

[0052] (4) Detection of the expression of mesenchymal markers in cells of each experimental group

[0053] Immunofluorescence staining was used to detect the expression of mesenchymal markers in cells of each experimental group.

[0054] The cells of control group 1, control group 2, and treatment group 2 were inoculated in a 12-well cell plate and placed on a slide. When the cell density on the slide reached 30%, DMSO was added to experimental group 1, and silybin and TGFβ1 were added to other treatment groups according to Table 1. After 48 hours of treatment, fixation, washing, perforation, blocking, incubation with primary antibody, washing, incubation with secondary antibody, washing, incubation with DAPI, washing, and sealing were performed to complete immunofluorescence staining. The images were observed and collected under a fluorescence microscope. The results are shown in Figure 4 The primary antibodies used were FN (1:1000) and αSMA (1:500).

[0055] like Figure 4 As shown, compared with the control group 2, the expression levels of αSMA and Fibronectin1 in the cells of treatment group 4 were significantly decreased, indicating that silybin can significantly downregulate the expression of mesenchymal markers.

[0056] When RPE cells undergo EMT, the expression level of intracellular mesenchymal markers is significantly upregulated, promoting the transformation of RPE cells into mesenchymal cells. The results of this experiment showed that silybin can prevent the morphology of ARPE-19 cells from transforming into mesenchymal cells, inhibit the mRNA expression of mesenchymal markers COL1A1, MMP2, Fibronectin1 and the protein expression of COL1A1, MMP2, Fibronectin1, N-cadherin and αSMA, indicating that silybin has the effect of inhibiting the EMT process of ARPE-19 cells.

[0057] Experimental Example 2 In vivo anti-PVR experiment of silybin

[0058] In order to verify whether silibinin has the effect of inhibiting proliferative vitreoretinopathy (PVR) in vivo, the anti-PVR effect of silibinin in vitro was evaluated through animal experiments. The specific scheme is as follows:

[0059] (1) Experimental materials and establishment of rat PVR model

[0060] The experimental subjects were 6-8 weeks old Long-Evans rats. There were 3 experimental groups in total, including control group, TGFβ1 group and TGFβ1+silybin group. The treatment of each group is shown in Table 2. There were 6 rats in the control group, 15 rats in the TGFβ1 group and 14 rats in the TGFβ1+silybin group. The animal treatment methods and experimental protocols were approved by the Animal Care and Use Committee of Sun Yat-sen University and strictly complied with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH).

[0061] Table 2 Treatment of each experimental group

[0062]

[0063] (2) Detection of rat fundus

[0064] The fundus of each rat was observed before injection and on days 7, 14, and 21 after injection, and fundus photographs were collected using a retinal imaging system. PVR was graded according to the vitreoretinal lesions of the rats. The rating was based on the Behar-Cohen rat PVR grading scale, as follows:

[0065] Grade 0: no proliferative reaction in the vitreous retina; Grade 1: proliferative reaction limited to the vitreous cavity; Grade 2: epiretinal membrane formation, accompanied by retinal wrinkles; Grade 3: white dense proliferative membrane covering the retina, accompanied by retinal wrinkles and localized retinal detachment, with or without localized posterior capsular cataract. The higher the grade, the more severe the PVR. Fundus photos and PVR rating results are shown in Figure 5 .

[0066] like Figure 5 As shown, vitreous opacity and preretinal proliferative membrane were observed in the TGFβ1 group on the 7th day after injection, while no obvious pathological changes were observed in the TGFβ1+silybin group; on the 14th day, dense preretinal proliferative membrane formation was observed in the TGFβ1 group accompanied by tractional shallow retinal detachment, while only loose preretinal proliferative membrane formation was found in the TGFβ1+silybin group; on the 21st day, obvious tractional retinal detachment was observed in the TGFβ1 group, while only local preretinal proliferative membrane formation was found in the TGFβ1+silybin group. According to statistics, the PVR levels of rats in the TGFβ1+silybin group were lower than those in the TGFβ1 group on the 7th, 14th and 21st days, and the differences between the groups were statistically significant (p<0.05).

[0067] (3) Detection of rat retinal morphology and expression levels of ocular mesenchymal markers

[0068] On the 21st day after injection, the rats were killed and the eyeballs were removed and made into paraffin sections. The paraffin sections were stained with HE, observed under an optical microscope and photographed. The paraffin sections were dewaxed and then immunofluorescence stained. The immunofluorescence staining steps were the same as those in item (4) of Experimental Example 1. The eyes were observed under a fluorescence microscope and photographed. The HE staining results are shown in Figure 6 -A, immunofluorescence staining results are shown in Figure 6 -B.

[0069] like Figure 6 As shown in the figure, on the 21st day after injection, the retina of the TGFβ1 group was shrunken and detached, and a dense proliferative membrane containing a large number of cellular components was visible on the surface, while the retina of the rats in the TGFβ1+silybin group was only slightly shrunken. Immunofluorescence staining of αSMA in the eyeball sections of rats on the 21st day after injection showed that αSMA was positively expressed in the proliferative membrane of the PVR in the TGFβ1 group, while there was less αSMA positive staining on the retinal surface in the TGFβ1+silybin group.

[0070] From the above results, it can be concluded that silybin can prevent the formation of epiretinal proliferative membrane in rats in vivo, prevent retinal shrinkage and tractional retinal detachment, and thus prevent the progression of PVR.

[0071] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. Application of silybin in the preparation of drugs for preventing proliferative vitreoretinopathy, It is characterized in that The proliferative vitreoretinopathy is caused by rhegmatogenous retinal detachment; The silybin includes silybin A and silybin B; The mass ratio of silybin A to silybin B in the silybin is silybin A:silybin B=1:

1.

2. The use according to claim 1, It is characterized in that The dosage form of the drug for preventing proliferative vitreoretinopathy is at least one of capsules, tablets, microcapsule preparations, injections, suppositories, sprays, ointments, gels, solutions, powders, lotions, tinctures, oils, creams and aerosols.

3. The use according to claim 1, It is characterized in that The drug for preventing proliferative vitreoretinopathy is used for vitreous cavity injection.

4. The use according to claim 3, It is characterized in that The dosage of the drug for preventing proliferative vitreoretinopathy for intravitreal injection is 100-200 μM silybin.

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