Application of MERTK inhibitor in preparation of medicine for treating pterygium

Abstract

The invention discloses an application of an MERTK inhibitor in preparation of a medicine for treating pterygium, the specific effect of the MERTK inhibitor in the process of inhibiting the occurrence and development of the pterygium is found for the first time, and the MERTK inhibitor has no obvious toxic or side effect on normal conjunctival tissues and can be independently or jointly used as a means for preventing and treating the pterygium to be developed. Compared with other medicine treatment, the MERTK inhibitor has higher targeting property on pterygium treatment and smaller toxic and side effects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a new application of MERTK inhibitors in the preparation of drugs for treating pterygium. Background Art

[0002] Pterygium is an abnormal proliferative ocular surface disease, manifested as a triangular pterygoid tissue extending from the bulbar conjunctiva to the cornea, which affects vision and appearance, and its global prevalence rate is 12%. Currently, it is considered that the pathogenesis of pterygium mainly includes ultraviolet irradiation, viral infection and genetic factors. Ultraviolet rays can damage the limbal stem cells with a barrier function, resulting in the invasion of conjunctival tissue and corneal damage. Ultraviolet rays can also trigger an oxidative stress cascade reaction, directly or indirectly destroying the integrity of DNA. The massive activation of human papillomavirus E6 and E7 genes will disrupt the normal function of p53 and promote the formation of pterygium. In addition, p53 gene mutations, mutations in the promoter encoding telomerase reverse transcriptase, and abnormalities in genes related to nicotinic acetylcholine receptors are closely related to the occurrence and development of pterygium. These genetic variations may lead to abnormal cell behaviors such as uncontrolled cell proliferation and blocked apoptosis, thus playing an important role in the pathogenesis of pterygium. Currently, surgery is still the preferred treatment method, mainly including bare sclera (BS), conjunctival autograft (CAT), limbal-conjunctival autograft (LAT), and amniotic membrane graft (AMT).

[0003] However, a systematic review of retrospective studies on the recurrence rate of primary pterygium surgery published between 1993 and 2022 showed that the global average recurrence rate of CAT was 7.61% (1.10% - 24.3%), that of LAT was 5.50% (0.60% - 18.40%), and that of AMT was 9.0% (3.60% - 29.40%). The results of this study indicate that, although there are certain differences in the recurrence rate among different surgical methods, none of them can achieve a radical cure for this disease, suggesting that further optimization of treatment strategies is needed to reduce the recurrence risk. Therefore, in order to reduce the recurrence risk of pterygium, adjuvant treatment is still required. Currently, commonly used drug and physical treatment methods include antimetabolites (such as mitomycin), cytotoxic drugs (such as 5-fluorouracil), anti-inflammatory drugs (such as glucocorticoids), anti-angiogenic drugs (such as ranibizumab and bevacizumab), and β-ray therapy. However, long-term use of cytotoxic and antimetabolite drugs may cause scleral and corneal damage, increasing the risk of scleral thinning and corneal ulcer. Radiotherapy has strict limitations on the irradiation dose, time, and area. If not handled properly, it may cause obliterative endarteritis or radiation cataract, and has high technical requirements for medical operations. In addition, ranibizumab and bevacizumab are expensive, and long-term use will impose a relatively large economic burden on patients.

[0004] Therefore, developing a safer and more effective postoperative anti-recurrence treatment strategy is of great significance for improving the clinical treatment effect of pterygium. MERTK is a member of the TAM receptor family, and its overexpression and dysfunction have been found to be key factors in the occurrence and development of various neoplastic and fibrotic diseases (including breast cancer, colorectal cancer, head and neck squamous cell carcinoma, idiopathic pulmonary fibrosis, etc.). UNC2250 is a highly effective and specific MERTK inhibitor with a half-maximal inhibitory concentration (IC50) of 1.7 nM, and its selectivity for MERTK is 160 times and 60 times that of Axl and Tyro3, respectively, which belong to the same TAM family. Studies have shown that UNC2250 can significantly inhibit the invasion and migration ability of lymphoma cells, enhance their sensitivity to anti-tumor drugs, and prolong the survival time of mice in the transplantation model. However, there is currently no study on the application of MERTK inhibitors in the treatment of pterygium. Summary of the Invention

[0005] Object of the Invention: The technical problem to be solved by the present invention is to provide a new drug that can be used to treat pterygium in view of the deficiencies of the prior art.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows: Use of MERTK inhibitor in the preparation of a medicament for treating pterygium. The present invention discovers for the first time the specific role of MERTK inhibitor in inhibiting the occurrence and development of pterygium, and it has no obvious toxic and side effects on normal conjunctival tissue. It can be developed as a means for preventing and treating pterygium alone or in combination with other drugs.

[0007] Therefore, the present invention not only provides a new application of MERTK inhibitor, but also provides a new therapeutic drug and treatment approach for the treatment of pterygium.

[0008] Further, the MERTK inhibitor is UNC2250, and its chemical formula is C 24 H 36 N 6 O 2 , and its CAS number is 1493694-70-4.

[0009] Further, the MERTK inhibitor can inhibit the migration ability and / or proliferation ability of pterygium fibroblasts.

[0010] Further, the MERTK inhibitor can reduce the expression levels of fibrosis-related proteins α-SMA and / or Fibronectin in pterygium fibroblasts.

[0011] Further, the MERTK inhibitor can reduce the proportion of G1-phase / S-phase cells, increase the proportion of G2-phase / M-phase cells, and block the cell growth cycle process of pterygium fibroblasts.

[0012] Further, the MERTK inhibitor can reduce the expression levels of cell cycle-related proteins CDK1, CCNB1, CCNE2, and CCNA2 in pterygium fibroblasts.

[0013] Further, the MERTK inhibitor can reduce the phosphorylation levels of AKT, PI3K, ERK, and p-38 in pterygium fibroblasts.

[0014] Further, the MERTK inhibitor is the only active ingredient in the medicament.

[0015] Further, the medicament includes a MERTK inhibitor and other active ingredients for treating or alleviating pterygium.

[0016] Furthermore, the present invention also claims the use of MERTK inhibitor in the preparation of a reagent for specifically targeting pterygium fibroblasts.

[0017] Compared with the prior art, the present invention has the following advantages: (1) The MERTK inhibitor of the present invention can specifically target the highly expressed molecule in pterygium fibroblasts, while the MERTK molecule is lowly expressed in normal conjunctival tissue cells and is not easily affected by the MERTK inhibitor. Therefore, compared with other drug treatments, the MERTK inhibitor has higher targeting and fewer side effects in the treatment of pterygium.

[0018] (2) The MERTK inhibitor of the present invention can widely inhibit various abnormal cell behaviors of pterygium fibroblasts. It can block the cell cycle of pterygium fibroblasts, inhibit the proliferation and migration abilities, and promote apoptosis, that is, it has a wide inhibitory effect on abnormal cell behaviors. Compared with other drug treatments, the therapeutic effect of the MERTK inhibitor is more significant. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The following further specifically describes the present invention in conjunction with the drawings and specific embodiments, and the above and / or other advantages of the present invention will become clearer.

[0020] Figure 1 is the chemical structural formula of the MERTK inhibitor UNC2250 of the present invention.

[0021] Figure 2 are the extraction and identification results of pterygium fibroblasts and normal conjunctival fibroblasts.

[0022] Figure 3 is the effect of the MERTK inhibitor on cell proliferation viability.

[0023] Figure 4 is the effect of the MERTK inhibitor on the migration ability of pterygium fibroblasts.

[0024] Figure 5 is the effect of the MERTK inhibitor on the fibrosis level of pterygium fibroblasts.

[0025] Figure 6 is the effect of the MERTK inhibitor on the apoptosis level of pterygium fibroblasts.

[0026] Figure 7 is the effect of the MERTK inhibitor on the proportion of proliferating cells in pterygium fibroblasts.

[0027] Figure 8 is the effect of the MERTK inhibitor on the cell growth cycle of pterygium fibroblasts.

[0028] Figure 9 is the effect of the MERTK inhibitor on the levels of cell growth cycle-related proteins in pterygium fibroblasts.

[0029] Figure 10Effect of MERTK inhibitor on the levels of cell growth-related signaling pathways in pterygium fibroblasts. Detailed implementation mode

[0030] The present invention can be better understood according to the following embodiments.

[0031] Example 1 1. Extraction of primary pterygium fibroblasts and normal conjunctival fibroblasts Take the surgically removed pterygium or normal conjunctival tissue, wash it 3 times with sterile PBS, cut the tissue into meat particles of 1-2 mm2 with an ophthalmic scissors, place them in a well plate pre-wetted with the culture medium, and add sufficient culture medium after the tissue adheres to the wall (usually 12-24 hours are required), and change the liquid every 2 days. Passage is carried out when the tissue crawls out to complete confluence (usually 10-14 days are required), and passages 4-9 are taken for subsequent experiments. The culture medium used for culturing primary pterygium fibroblasts and primary conjunctival fibroblasts is F12 medium (containing 10% fetal bovine serum, 1% penicillin-streptomycin), and the culture environment is 5% CO2, 37 °C. The digestion condition is 0.25% trypsin, 1 minute at 37 °C, and the digestion is terminated with complete medium and resuspended, centrifuged at 1000 rpm for 5 minutes, and passaged at a ratio of 1:2. The specific MERTK inhibitor is UNC2250 (S7342, purity: 99.43%, Selleck, USA). The 1 μM and 2 μM MERTK inhibitor treatment groups are used as the low-concentration experimental group and the high-concentration experimental group respectively, and the solvent group is used as the control group.

[0032] Figure 1 Shown is the chemical structure of the MERTK-specific inhibitor UNC2250.

[0033] 2. Protein extraction and Western blotting (1) Sample lysis: Taking a 6-well plate as an example, wash it 2 times with pre-cooled PBS and aspirate the PBS. Mix RIPA lysis buffer and PMSF protease inhibitor evenly at a ratio of 100:1, add 100 μL to each well, and lyse on ice for 10 min, then use a cell scraper to collect the sample into a 1.5 mL centrifuge tube.

[0034] (2) Protein extraction: Place the sample in a centrifuge pre-cooled to 4 °C, centrifuge at 13000 g for 20 min, and the supernatant after centrifugation is the extracted protein. Carefully transfer the supernatant to a new centrifuge tube. Prepare the protein standard according to the BCA protein quantification kit instruction manual. Incubate the BCA reagent with the sample to be measured at 37 °C for 30 min, then use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance of the protein standard and the protein sample, make a concentration standard curve, and calculate the concentration of the protein sample.

[0035] (3) Protein denaturation: Add 5× protein loading buffer to the protein sample and heat it at 50 °C for 10 min to denature the protein. After denaturation, the sample is aliquoted and stored at -80 °C in the refrigerator.

[0036] (4) Electrophoresis: Prepare the SDS-PAGE gel according to the instructions of the SDS-PAGE Gel Rapid Preparation Kit. Place the SDS-PAGE gel in the electrophoresis tank and add 1 L of electrophoresis buffer. The amount of protein loaded is 20 - 40 μg, and 5 μL of protein Marker is added to the wells on both sides of the sample to mark the protein size. Electrophoresis is carried out at a constant voltage. First, use 80 V for electrophoresis for 30 min. After the sample enters the separation gel, adjust the voltage to 120 V and continue electrophoresis for 60 min.

[0037] (7) Membrane transfer: After electrophoresis, take out the SDS-PAGE gel and place it in the pre-cooled membrane transfer solution. Cut the PVDF membrane with a pore size of 0.22 μm into an appropriate size and activate it with methanol for 1 min. Moisten the filter paper and sponge with the membrane transfer solution in advance and arrange them in the order: black side of the electrophoresis clip - sponge - filter paper - gel - PVDF membrane - filter paper - sponge - white side of the electrophoresis clip. After removing the air bubbles between each layer, fix the electrophoresis clip and put it into the membrane transfer tank, and transfer the membrane at a constant current of 250 mA for 30 - 120 min.

[0038] (8) Blocking: After membrane transfer, place the PVDF membrane in the prepared 5% skim milk powder or 5% BSA and gently shake it at room temperature for blocking for 1 - 2 h. After blocking, wash it 3 times with TBST buffer on a high-speed shaker, 5 min each time.

[0039] (9) Primary antibody incubation: Prepare the primary antibody (1:1000) with the primary antibody diluent, immerse the target band in the corresponding primary antibody, and incubate it overnight at 4 °C. After incubation, wash it 3 times with TBST buffer on a high-speed shaker, 10 min each time.

[0040] (10) Secondary antibody incubation: Dilute the goat anti-rabbit-HRP antibody and goat anti-mouse-HRP antibody (1:10000), immerse the target band in the corresponding secondary antibody, and incubate it at room temperature for 1 h. After incubation, wash it 3 times with TBST buffer on a high-speed shaker, 10 min each time.

[0041] (11) ECL development: Prepare the ECL chemiluminescent solution into a working solution and use it immediately. Immerse the protein band in the ECL working solution and incubate it in the dark for about 1 min, then blot off the excess working solution with absorbent paper. Use a fully automatic chemiluminescent imaging analysis system to collect images and detect the protein expression level.

[0042] Figure 2Protein immunoblotting showed that both types of fibroblasts expressed vimentin and did not express keratin, which was consistent with the characteristics of fibroblasts. Moreover, the level of MERTK in fibroblasts of pterygium was significantly higher than that in fibroblasts of normal conjunctival tissue. Figure 5 It was shown that after treatment with MERTK inhibitor, the fibrosis levels (α-SMA, FN) of fibroblasts in pterygium were significantly reduced. Figure 9 It was shown that after treatment with MERTK inhibitor, the levels of cell cycle-related proteins (including CDK1, CCNA2, CCNB1, CCNE2) in fibroblasts of pterygium were significantly down-regulated. Figure 10 It was shown that there were no obvious changes in the total protein levels of AKT, PI3K, ERK, and P38 in fibroblasts of pterygium in the low- and high-concentration groups, while the phosphorylation levels were significantly down-regulated.

[0043] 3. Cell viability assay The CCK-8 (Cell counting kit-8) method was used to detect cell viability. Pterygium fibroblasts and normal conjunctival fibroblasts in the logarithmic growth phase were taken, digested and centrifuged, and then resuspended into cell suspensions. The cell density was adjusted to 4×104 / mL, and 100 μL was inoculated into each well of a 96-well plate, that is, 4×103 cells per well. Each treatment group was repeated 6 times. After treatment, the cells in each well were replaced with 100 µL of fresh medium containing 10 μL of CCK-8 reagent and incubated in an incubator at 37 °C for 2 hours. Finally, the OD value of each well was detected by an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 450 nm.

[0044] Figure 3 It was shown that low-concentration (≥ 0.5 μM) MERTK inhibitor could significantly inhibit the proliferation viability of pterygium fibroblasts (inhibition rate exceeded 20%). Low- and medium-concentration (0.5 μM - 2 μM) MERTK inhibitor had no obvious effect on the proliferation viability of normal conjunctival fibroblasts. High-concentration (≥ 2.5 μM) MERTK inhibitor had a significant effect on the proliferation ability of both types of fibroblasts (inhibition rate exceeded 30%).

[0045] Example 2 Scratch assay 5×104 pterygium fibroblasts were inoculated into a 24-well cell culture plate and allowed to grow until they reached 100% confluence to form a monolayer of cells. The cells were starved in serum-free medium for 24 hours. A sterile 200 µL pipette tip was used to gently scratch the monolayer of cells. After scratching, the medium was removed and the cells were washed with PBS to remove cell debris. Media containing different concentrations of MERTK inhibitor were applied, and the cells were cultured in a 5% CO 2 , 37 °C environment for continued culture. Photos were taken of the scratched area at 0 hours, 24 hours, 48 hours, and 72 hours.

[0046] The scratch assay showed that low and high concentrations of the MERTK inhibitor could inhibit the low migratory ability of pterygium fibroblasts at each time point ( Figure 4 ).

[0047] Example 3 Transwell assay Pterygium fibroblasts were resuspended with low serum culture using different drug concentrations. 4 × 10 4 cells were added to the upper layer of each Transwell chamber, and 500 μL of complete medium containing the corresponding drug concentration was added to the lower layer. After incubation for 24 hours and 48 hours, the Transwell chambers were fixed with 4% paraformaldehyde at room temperature for 10 minutes, stained with crystal violet for 30 minutes, the cells in the upper chamber were removed with a cotton swab, the remaining cells in the upper chamber were washed with PBS, and the number of stained cells was counted under a microscope.

[0048] The Transwell assay showed that the scratch assay showed that low and high concentrations of the MERTK inhibitor could inhibit the low migratory ability of pterygium fibroblasts at each time point ( Figure 5 ).

[0049] Example 4 Apoptosis flow cytometry Pterygium fibroblasts were pretreated with different concentrations of the drug for 48 hours, digested with trypsin to obtain a cell suspension, washed twice with PBS, stained with Annexin V-FITC / PI, and detected using a flow cytometer. FITC and PE double-positive cells were late apoptotic cells.

[0050] Figure 6 It was shown that the apoptosis ratio of pterygium fibroblasts in the low and high concentration groups increased significantly.

[0051] Example 5 Cell cycle detection Pterygium fibroblasts were pretreated with different concentrations of the drug for 48 hours, digested with trypsin without EDTA, and centrifuged into a 1.5 mL EP tube. Fixed with pre-cooled 70% ethanol at -20 °C for 12 hours. Centrifuged at 3000 rpm for 10 minutes, washed twice with PBS, stained with 500 µl of PI / RNase complex solution, incubated for 15 minutes at room temperature in the dark, and detected using a flow cytometer. Cells with 2-fold fluorescence intensity were G1-phase cells, cells with 4-fold fluorescence intensity were G2-phase cells, and cells with 2-4-fold fluorescence intensity were S-phase cells.

[0052] Figure 8 It was shown that the G1 / S phase ratio of pterygium fibroblasts in the low and high concentration groups decreased significantly, and the G2 / M phase ratio increased significantly.

[0053] Example 6 EdU proliferation assay Seed 3×103 cells per well in a 96-well plate and pretreat pterygium fibroblasts with different concentrations of the drug for 48 hours. Add 100 μL of EdU reagent (10 μM) to each well and incubate at 37 °C for 12 hours to label proliferating cells. Fix with 4% paraformaldehyde at room temperature for 15 minutes and permeabilize with 0.3% Triton X-100 at room temperature for 15 minutes. Add click reaction reagent and incubate in the dark at room temperature for 30 minutes. Stain cell nuclei with Hoechst 33342. Take cell pictures using an inverted fluorescence microscope.

[0054] Figure 7 It is shown that the number of EdU-stained positive pterygium fibroblasts in the low- and high-concentration groups is significantly reduced, and the proportion of cells in the proliferative phase is significantly reduced.

[0055] Example 7 RNA extraction and real-time fluorescence quantitative reverse transcription polymerase chain reaction (1) Cell lysis: After discarding the culture medium, add an appropriate amount of pre-cooled PBS to wash the cells and discard the PBS. Taking a 6-well plate as an example, add 1 mL of TRIzol reagent to each well, let it stand for 5 min, then pipette to blow and transfer the sample to a 1.5 mL centrifuge tube.

[0056] (2) RNA extraction: Add 200 μL of chloroform to the lysed sample, shake vigorously for 15 s, then let it stand on ice for 5 min, and then centrifuge the sample at 12000 g at 4 °C for 15 min. Carefully take out the centrifuge tube, transfer the upper aqueous phase to a new centrifuge tube, add an equal volume of pre-cooled isopropanol, invert and mix well, let it stand on ice for 10 min, and then centrifuge the sample at 12000 g at 4 °C for 10 min. At this time, a white precipitate can be seen. Discard the supernatant, add 1 mL of 75% ethanol to wash the precipitate, let it stand at room temperature for 5 min, and then centrifuge the sample at 12000 g at 4 °C for 5 min, and discard the supernatant.

[0057] (3) RNA concentration determination: In a clean environment, dry the RNA precipitate in the open air at room temperature for 2 - 5 min, and add an appropriate amount of DEPC water to dissolve the precipitate. Take a small amount of the sample and use a Nanodrop ultraviolet micro-spectrophotometer to detect the concentration and purity. Store the remaining unused RNA at -80 °C.

[0058] (4)RNA reverse transcription to synthesize cDNA: Take 1 μg of total RNA from each sample and place it in an enzyme-free polymerase chain reaction tube. Synthesize cDNA according to the instructions of HiScript II Q Select RT SuperMix. The specific steps are as follows: Make up the volume of 1 μg of total RNA to 12 μL with DEPC water, add 4 μL of 4×gDNA Wiper Mix, and react in a PCR instrument at 42 °C for 2 min to remove genomic DNA. After the reaction is completed, add 4 μL of 5×HiScript II Q Select RT SuperMix, and perform reverse transcription in a PCR instrument according to the program of 37 °C for 15 min, 85 °C for 5 s, and store at 4 °C. The finally obtained cDNA is stored at -80 °C.

[0059] (5)Real-time fluorescence quantitative polymerase chain reaction: Dilute the cDNA obtained after reverse transcription 10-fold with DEPC water. Set 3 replicate wells for each sample, use β-actin as the internal reference gene, and calculate the relative mRNA expression level of the gene by the 2-ΔΔCT method. The reaction system is shown in Table 1.

[0060] Table 1

[0061] Figure 9 It is shown that the mRNA expression levels of cell cycle-related genes (including CDK1, CCNA2, CCNB1, CCNE2) in pterygium fibroblasts in the low- and high-concentration groups are significantly reduced.

[0062] Based on the above experimental results, it can be fully demonstrated that MERTK inhibitors slow down the occurrence and development of pterygium by blocking the cell cycle of pterygium fibroblasts, inhibiting the proliferation and migration ability, and promoting apoptosis. Therefore, we believe that MERTK inhibitors can be used as a new and important drug for the clinical prevention and treatment of pterygium, and have potential clinical application value in the prevention and treatment of pterygium.

[0063] The present invention provides an idea and method for the application of an MERTK inhibitor in the preparation of a drug for treating pterygium. There are many methods and ways to specifically implement this technical solution. The above is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention. Each component not clearly defined in this embodiment can be implemented by existing technologies.

Claims

1. Application of MERTK inhibitors in the preparation of drugs for the treatment of pterygium.

2. The use according to claim 1, characterized in that: The MERTK inhibitor is UNC2250.

3. The use according to claim 1 or 2, characterized in that: The MERTK inhibitor can inhibit the migration ability and / or proliferation ability of pterygium fibroblasts.

4. The use according to claim 1 or 2, characterized in that: The MERTK inhibitor can reduce the expression level of fibrosis-related proteins α-SMA and / or Fibronectin in pterygium fibroblasts.

5. The use according to claim 1 or 2, characterized in that: The MERTK inhibitor can reduce the ratio of G1 phase / S phase cells, increase the ratio of G2 phase / M phase cells, and block the cell growth cycle process of pterygium fibroblasts.

6. The use according to claim 1 or 2, characterized in that: The MERTK inhibitor can reduce the expression levels of cell cycle-related proteins CDK1, CCNB1, CCNE2 and CCNA2 in pterygium fibroblasts.

7. The use according to claim 1 or 2, characterized in that: The MERTK inhibitor can reduce the phosphorylation levels of AKT, PI3K, ERK and p-38 in pterygium fibroblasts.

8. The use according to claim 1 or 2, characterized in that: The MERTK inhibitor is the only active ingredient in the drug.

9. The use according to claim 1 or 2, characterized in that: The drug includes a MERTK inhibitor and other active ingredients for treating or alleviating pterygium.

10. Use of a MERTK inhibitor in the preparation of a reagent expressing specific targeting pterygium fibroblasts.

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