CN111617066B - Application of Chalcomoracin in the preparation of drugs for the treatment of proliferative vitreoretinopathy - Google Patents

Abstract

The invention discloses the application of Chalcomoracin and its homologues in preparing medicines for treating and preventing proliferative vitreoretinopathy. Chalcomoracin and its homologues can inhibit vitreous-induced AKT activation and p53 decrease, block vitreous-stimulated ARPE‑19 cell proliferation, migration and contraction, and have therapeutic and preventive effects on proliferative vitreoretinopathy.

Description

Application of Chalcomoracin in the preparation of drugs for the treatment of proliferative vitreoretinopathy

technical field

The invention belongs to the field of biomedicine, and particularly relates to the application of Chalcomoracin and its homologues in preparing medicines for treating and preventing proliferative vitreoretinopathy.

Background technique

Proliferative vitreoretinopathy (PVR) is a common complication of rhegmatogenous retinal detachment, and it is also one of the main reasons for the failure of rhegmatogenous retinal detachment surgery and the poor postoperative visual recovery. The pathogenesis of PVR is divided into several stages: 1) Cell migration, mainly RPE and glial cells. 2) Proliferation of migrating cells; 3) Development of membranes; 4) Contraction of cell membranes; 5) Production of extracellular collagen; 6) Formation of fixed folds in the retina. PVR is characterized by retinal detachment and membrane growth on both sides of the posterior vitreous membrane. Retinal pigment epithelium (RPE) plays an important role in the development and progression of proliferative vitreoretinopathy. When the retina is detached due to contraction, retinal cells (such as RPE) form the epiretinal or submembrane membrane and undergo a series of responses, including proliferation and migration. So far, there is no effective drug that can prevent the formation of PVR.

Chalocomoracin is the major secondary metabolite produced by fungal-infected mulberry leaves and protects the leaves by inhibiting the fungus. Chalocomoracin has been reported to have broad biological activity against rhinovirus, methicillin-resistant Staphylococcus aureus, and human cancer cell lines, but its effect on PVR has not been investigated.

SUMMARY OF THE INVENTION

The invention provides the application of chalcomoracin and its homologues in the preparation of proliferative vitreoretinopathy drugs, which can inhibit the activation of AKT and the decrease of p53 induced by the vitreous body, and can block the proliferation, migration and contraction of ARPE-19 cells stimulated by the vitreous body. Role in the treatment and prevention of proliferative vitreoretinopathy.

The present invention adopts the following technical solutions to realize:

Application of Chalcomoracin and its homologues in the preparation of medicaments for the treatment and prevention of proliferative vitreoretinopathy.

In the above-mentioned technical scheme, preferably, Chalcomoracin and its homologues are prepared through conventional preparation process, alone or in combination with other medicines to prepare medicines of various dosage forms that can be used clinically, including injections, tablets, capsules, soft capsules, Microcapsules, nano-formulations, granules, films, suppositories, aerosols and other dosage forms.

The beneficial effects of the present invention are:

The present invention provides a pharmaceutical raw material or a latent drug for the treatment and prevention of proliferative vitreoretinopathy, which can inhibit the activation of AKT induced by the vitreous body and the decrease of p53, and can block the proliferation, migration and contraction of ARPE-19 cells stimulated by the vitreous body. This mechanism plays a role and has good development potential.

Description of drawings

Figure 1 Chalcomoracin on vitreous-induced AKT activation and p53 reduction, (A) Western blot analysis, (B) p-Akt, intensities of Akt and p53 bands (*p<0.05, **p<0.01, ***p <0.001);

Figure 2 The effect of Chalcomoracin on vitreous-induced cell proliferation (*** indicates p<0.001);

Figure 3 The effect of Chalcomoracin on vitreous-induced scratch repair, (A) photo of scratch repair, (B) cell migration rate (* means p<0.05, *** means p<0.001);

Figure 4 The effect of Chalcomoracin on collagen contraction (* indicates p<0.05).

Detailed ways

The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

The application of Chalcomoracin and its homologues of the present invention in the preparation of medicines for the treatment and prevention of proliferative vitreoretinopathy.

The present invention also provides Chalcomoracin and its homologues alone or in combination with other medicines to prepare medicines in various dosage forms that can be used clinically, including injections, tablets, capsules, soft capsules, microcapsules, nano-formulations, granules, Films, suppositories, aerosols and other dosage forms.

In order to make the above objects, features and advantages of the present invention more obvious and easy to understand, the technical solutions of the present invention are further described below with reference to the embodiments. However, the present invention is not limited to the listed embodiments, but also includes any other known modifications within the scope of the claims of the present invention.

Example 1 Chalcomoracin inhibits vitreous-induced AKT activation and p53 decrease

After resuscitation of ARPE-19 cells, they were placed in a 24-well plate in DMEM/F12 medium containing 10% fetal bovine serum, and cultured in a 37°C, 5% CO ₂ incubator. After the cells were confluent to 90%, the cells were serum-starved for 24 hours and then treated with drugs (PVR rabbit vitreous (RV, diluted 1:3 with DMEM/F12 medium), medium containing RV and 5 μM Chalcomoracin (CMR) for 8 hours. Wash twice with pre-cooled phosphate buffered saline (PBS), add lysate and let stand on ice for 15min, collect lysate, centrifuge at 12000r/m for 15min at 4°C. After BCA quantification, add a certain volume of loading buffer and boil Protein samples were obtained in 15 min. Separated by 10% SDS-polyacrylamide gel electrophoresis. The proteins in the gel were then transferred to a polyvinylidene fluoride membrane and subjected to Western blot analysis with the desired antibody. Repeated at least three times independent experiment.

Western blot analysis showed that vitreous (RV, diluted 1:3 in DMEM/F12 medium) could significantly promote AKT activation in ARPE-19 cells and also inhibit p53 expression, and 5 μM Chalcomoracin could significantly block vitreous-induced AKT activation ( 1A and 1B), and inhibited the inhibitory effect of the vitreous on p53 expression. These data suggest that the pathogenesis of Chalcomoracin preventing PVR focuses on inhibition of AKT activation and inhibition of P53 reduction.

Example 2 Cell Proliferation Experiment

After resuscitation of ARPE-19 cells, they were placed in a 24-well plate in DMEM/F12 medium containing 10% fetal bovine serum, and cultured in a 37°C, 5% CO ₂ incubator. After ARPE-19 reached about 90% confluence, they were digested with 0.5% Tripsin-EDTA, counted, seeded in wells of 24-well plates at a density of ³ x 104 cells/well, and plated in 10% fetal bovine Serum DMEM/F12 medium, after the cells were plated, treated with 0.5ml DMEM(-) or RV supplemented with 5μM Chalcomoracin for 48h, trypsinize the cells, and count the cells under a light microscope by hemocytometry, taking three times The experimental results are processed for data processing. RV stimulated ARPE-19 proliferation (1.6±0.2-fold), while Chalcomoracin completely abolished RV-mediated cell proliferation, see Figure 2.

Example 3 Cell Migration Experiment

After resuscitation of ARPE-19 cells, they were placed in a 24-well plate of DMEM/F12 medium containing 10% fetal bovine serum, and cultured in a 37°C, 5% _CO2 incubator. When ARPE-19 has grown to complete confluence, a "one" scratch is made by scraping with an autoclaved 200 μl pipette tip. Washed 3 times with PBS, the initial scratch width was photographed, then treated with RV (1:3 dilution in DMEM/F12) containing 0, 2.5, 5, 10 μM halcomoracin, scratches were photographed again after 24 hours, and then imaged using Image J and Adobe Photoshop CS4 software were used to analyze the data, and three experimental results were taken for data processing. The results of the scratch healing assay are shown in Figure 3, indicating that RV stimulated ARPE-19 migration (Figure 3A), while Chalcomoracin significantly inhibited RV-induced cell migration in a dose-dependent manner (Figure 3B).

Example 4 Collagen Contraction Experiment

After resuscitation of ARPE-19 cells, they were placed in a 24-well plate of DMEM/F12 medium containing 10% fetal bovine serum, and cultured in a 37°C, 5% _CO2 incubator. When the cells reached 90% confluence, they were plated with The density of 1 x 10 ⁶ was resuspended in 1.5 mg/ml neutralized PureCol type I bovine collagen solution (pH 7.2, ice bath), and after gentle mixing, the collagen gels and cells were transferred to 5 mg/ml In a 24-well plate pre-incubated overnight in bovine serum albumin (BSA)/PBS, the mixture of cells and gel in the plate was incubated at 37°C for 90 min to solidify the collagen. Subsequently, 0.5 ml of DMEM/F12, DMEM/F12 containing 5 μM halcomoracin, RV (1:3 dilution in DMEM/F12), 5 μM halcomoracin and RV (1:3 dilution in DMEM/F12) were added, and cultured for 48 h. , take a picture, and measure the gel diameter, calculate the gel area using the ^formula 3.14 × r, where r is the radius of the gel. The results are shown in Figure 4. The vitreous promotes ARPE-19 contraction, while Chalcomoracin blocks the vitreous-induced contraction.

It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be Modifications or equivalent replacements, without departing from the spirit and scope of the technical solutions of the present invention, should all be included in the scope of the claims of the present invention.

Claims (2)

1. Application of Chalcomoracin as the only active ingredient in the preparation of a drug for the treatment of proliferative vitreoretinopathy. 2 . The application of Chalcomoracin according to claim 1 as the sole active ingredient in the preparation of a medicine for the treatment of proliferative vitreoretinopathy, wherein the medicine is an oral preparation, an injection, an inhalation preparation or an external preparation.

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