阅读说明：本技术 一种视网膜保护剂、视网膜保护方法以及保护剂的应用 (Retina protective agent, retina protective method and application of protective agent ) 是由 白洁 张永明 杨帆 于 2021-08-02 设计创作，主要内容包括：本发明公开了一种视网膜保护剂、视网膜保护方法以及保护剂的应用,属于视网膜领域,一种视网膜保护剂,主要用于对糖尿病患者引起的视网膜损伤进行改善,相比于传统的抗VEGF药物和激素类药物对糖尿病视网膜病变的治疗,不易出现治疗终止,病变复发的问题,且本保护剂安全无毒,具有抑制细胞凋亡、抑制氧化损伤、延缓组织衰老等多重功效,市面上的价格也更低廉。(The invention discloses a retina protective agent, a retina protection method and application of the protective agent, belongs to the field of retinas, and the retina protective agent is mainly used for improving retina injury caused by diabetic patients, is less prone to the problems of treatment termination and recurrence compared with the traditional treatment of diabetic retinopathy by anti-VEGF (vascular endothelial growth factor) medicines and hormone medicines, is safe and non-toxic, has multiple effects of inhibiting apoptosis, inhibiting oxidative damage, delaying tissue aging and the like, and is lower in price in the market.)

1. A retinal protective agent characterized by: the protective agent comprises growth hormone releasing peptide, and the protective agent is cultured in a cell culture solution when in use.

2. The agent according to claim 1, wherein: the components of the cell culture solution comprise glucose, amino acids, inorganic salts, vitamins and animal serum.

3. The agent according to claim 1, wherein: the ghrelin was purchased from Sigma Chemical.

4. A method of retinal protection according to claim 1 wherein: use of a retinoprotective agent as in any of claims 1-3.

5. Use of a retinal protective agent according to claim 1 in the manufacture of a medicament for the protection of photoreceptor apoptosis in diabetic retinopathy.

6. Use of a retinal protective agent according to claim 1 in the manufacture of a medicament for protecting photoreceptor cell viability in diabetic retinopathy.

7. Use of a retinoprotective agent in the manufacture of a medicament for the protection of the thickness of the retinal nerve fiber layer in diabetic retinopathy, in accordance with claim 1.

8. The use of a retinoprotective agent in the preparation of a medicament for improving the number of TUNEL staining positive cells in the retinal photoreceptor layer in diabetic retinopathy according to claim 1.

9. Use of a retinal protective agent according to claim 1 in the manufacture of a medicament for improving nuclear and cytoplasmic structures in diabetic retinopathy.

10. The use of a retinal protective agent according to claim 1 in the manufacture of a medicament for the inhibition of diabetic macular edema.

Technical Field

The invention relates to the field of retinas, in particular to a retina protective agent, a retina protective method and application of the protective agent.

Background

The long-term hyperglycemia and changes in blood composition in diabetic patients disrupt the blood-retinal barrier, perivascular necrosis of the retinal capillaries, endothelial dysfunction, resulting in leakage of fluid components in the blood vessels into the retinal tissue spaces, causing a series of changes in the retinal tissue: hemorrhage, edema, effusion, ischemia, etc.

Although anti-VEGF drugs and hormone drugs have been used for the treatment of diabetic retinopathy in recent years, the therapeutic targets are mainly directed to diabetic macular edema, and long-term continuous administration is required, the vision can be improved in stages during the treatment, once the treatment is stopped, macular edema is liable to recur, and this therapeutic method cannot fundamentally solve the problem of diabetic retinal damage. Therefore, exploring the mechanism of molecular biological changes in the early stages of diabetic retinopathy and finding potential therapeutic approaches are of great clinical significance.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to provide a retina protective agent, which contains a growth hormone releasing peptide component inside, can protect and improve the retina injury possibly caused by a diabetic, is not easy to cause the problems of treatment termination and disease recurrence compared with the traditional treatment of diabetic retinopathy by anti-VEGF medicines and hormone medicines, and has lower market price;

the second purpose is to provide a retina protection method, which uses the retina protective agent to protect the retina, is safe and nontoxic, and has multiple effects of inhibiting apoptosis, inhibiting oxidative damage, delaying tissue aging and the like;

the third purpose is to provide an application of the retina protective agent, and the retina protective agent can be applied to the preparation of drugs for protecting the apoptosis of photoreceptor cells in the diabetic retinopathy, the preparation of drugs for protecting the vitality of the photoreceptor cells in the diabetic retinopathy, the preparation of drugs for protecting the thickness of retinal nerve fiber layers in the diabetic retinopathy, the preparation of drugs for improving the number of TUNEL staining positive cells in the retinal photoreceptor cell layers in the diabetic retinopathy, the preparation of drugs for improving the structures of cell nucleus and cell cytoplasm in the diabetic retinopathy, the preparation of drugs for inhibiting diabetic macular edema and the like.

A retinal protective agent comprising ghrelin, wherein the agent is administered in a cell culture.

Further, the components of the cell culture solution comprise glucose, amino acids, inorganic salts, vitamins and animal serum.

Further, the ghrelin was purchased from Sigma Chemical (st. louis, MO, USA).

Further, the protective agent is applied to ocular tissue.

Further, the ocular tissue includes one or more of a lens, a trabecular meshwork, and an optic nerve.

A method of protecting retina by using the above-mentioned retina protecting agent.

An application of retina protective agent in preparing medicine for preventing the apoptosis of photosensitive cells in diabetic retinopathy is disclosed.

An application of retina protective agent in preparing medicine for protecting the activity of photosensitive cell in diabetic retinopathy is disclosed.

Use of a retinal protective agent in the manufacture of a medicament for protecting the thickness of retinal nerve fibre layers in diabetic retinopathy.

Use of a retinal protective agent in the preparation of a medicament for improving the number of TUNEL staining positive cells in a retinal photoreceptor layer in diabetic retinopathy.

Use of a retinal protective agent in the preparation of a medicament for improving nuclear and cytoplasmic structures in diabetic retinopathy.

An application of retina protective agent in preparing medicine for inhibiting diabetic macular edema is disclosed.

Compared with the prior art, the invention has the advantages that:

the retina protective agent of the scheme contains a growth hormone releasing peptide (Ghrelin) component, is mainly used for protecting and improving retina injury possibly caused by a diabetic patient, is difficult to have the problems of treatment termination and pathological change recurrence compared with the traditional treatment of the diabetic retinopathy by a VEGF resistant medicament and a hormone medicament, is safe and non-toxic, has multiple effects of inhibiting apoptosis, inhibiting oxidative damage, delaying tissue aging and the like, and is lower in market price.

Drawings

FIG. 1 is a retinal tissue map of a control group of the present invention;

FIG. 2 is a diagram of a model set retinal tissue of the present invention;

FIG. 3 is a graph of a ghrelin group retinal tissue of the present invention;

FIG. 4 is a graph showing TUNEL staining results of a control group of retinas according to the present invention;

FIG. 5 is a graph showing TUNEL staining results of a model group retina according to the present invention;

FIG. 6 is a graph showing the results of TUNEL staining of retinas of the ghrelin group according to the present invention;

FIG. 7 is a transmission electron microscope image of the retinal pigment epithelium layer of the control group of the present invention;

FIG. 8 is a transmission electron microscope image of the retinal pigment epithelium layer of the model group of the present invention;

FIG. 9 is a transmission electron microscope image of the retinal pigment epithelium layer of the ghrelin group according to the present invention;

FIG. 10 is a bar graph of pathological changes (× 200) of rat retina tissue according to the present invention.

Detailed Description

The method takes high-sugar induced retinal pigment epithelial cells and db/db mice as research objects, and observes the change rule of retinal pigment epithelial cell NLRP3 inflammation body activation and inflammatory factor release and the retinal damage condition after the Ghrelin is given in vitro, and clarifies a new mechanism for alleviating diabetic retinal damage by the Ghrelin, thereby providing a new theoretical basis for the prevention and treatment of diabetic retinopathy by the Ghrelin.

Example 1 (study of the protective effects of exogenous Ghrelin on retinal tissue in db/db mice):

the method comprises the following steps:

the method comprises the following steps: grouping and processing method of type II diabetes mice db/db: and (3) taking a db/db mouse with the age of 10 weeks, and identifying whether the diabetes model succeeds or not by using a method for measuring blood sugar by using a rat tail artery (the blood sugar is more than or equal to 16.6 mmol/L). Animal grouping: control, model and Ghrelin groups. Ghrelin group: the micro-pump was placed subcutaneously beginning the last two weeks in the model group and pumping Ghrelin (5. mu.g/kg/d) was continued for two weeks. .

Step two: HE staining examined retinal structure: dewaxing retina tissue with xylene, preparing paraffin section, dehydrating with gradient alcohol, washing with distilled water repeatedly, soaking in hematoxylin staining solution for 10 min, separating with ammonia water and acid water, soaking in gradient alcohol solution for 10 min, HE staining for 30 s, sealing, and taking picture.

Step three: observing retinal tissues by an electron microscope: taking out retinal tissue from glutaraldehyde fixation solution, washing with distilled water repeatedly, and washing with osmium tetroxide (OsO)₄) Fixing for 2 h, dehydrating with gradient alcohol, embedding with epoxy resin after dyeing, preparing ultrathin sections, observing rat retina tissues by a transmission electron microscope and taking pictures.

Step four: detecting Ghrelin and receptor expression: detecting Ghrelin expression in the room water by high performance liquid chromatography; detecting the expression of Ghrelin and GHSR-1a in retinal tissues by RT-PCR and Western blot method.

Step five: detecting the expression and activation of an inflammation body of retinal tissue NLRP 3: preparing retina tissue into a frozen section, and detecting the expression of NLRP3 inflammation bodies in the retina tissue and RPE cells by adopting an immunofluorescence method. The expression of ICAM-1 and TNF-alpha factors is detected by an ELISA method, and the expression condition of NLRP3 inflammasome proteins (NLRP 3 and pro-caspase-1) in retinal tissues is detected by a western blot technology.

Step six: detecting P in retinal tissue₂X₇R expresses: taking retinal tissue, and detecting P in the retinal tissue by using real-time PCR method₂X₇And R is expressed.

Example 2 (HE staining for retinal histopathological morphological changes):

3 rats were randomly selected for each group, and after removing the left eye, part of the retinal tissue was fixed with 4% paraformaldehyde for 2 hours, 5 μm retinal sections were placed on a glass slide, stained with H & E after gradient alcohol washing, and images were taken with a light mirror (200X).

S pathological changes in retinal tissue:

as shown in fig. 1-3, the control group of fig. 1 had intact retinal tissue and a clear structure; FIG. 2 is a graph showing that the thickness of the retina of the model group is obviously reduced, and the arrangement of cells in the inner nuclear layer and the outer nuclear layer is loose; FIG. 3 the ghrelin group shows increased retinal thickness and relatively regular structure compared to the model group.

Example 3 (TUNEL staining to detect apoptosis in rat retina):

the retina slices are soaked and washed twice by dimethylbenzene after being dewaxed, TUNEL reaction mixed liquor is added after alcohol gradient dehydration, the retina slices are incubated for 2 hours in a wet box, the spin-dried slices are mixed with DAB color developing solution, the cells are observed to be brownish yellow under a microscope and are TUNEL positive, the slices are washed by phosphate buffer solution to stop color development, neutral gum is sealed, and images are shot by a light mirror (200 x).

Retinal TUNEL staining results:

as shown in FIGS. 4-6, dark brown TUNEL staining positive cells were occasionally observed in the photoreceptor cell layer of rat retina in the control group of FIG. 4. Figure 5 model group TUNEL staining positive cells were significantly increased. FIG. 6ghrelin dry prognosis, reduction in the number of cells positive for TUNEL staining.

Example 4 (transmission electron microscopy of retinas):

3 rats in each group were taken, retinal tissues were fixed with 2% glutaraldehyde for 24 h after removal of the left eye, stained with a uranium acetate solution, dehydrated with an ethanol-acetone gradient solution, and stained with lead citrate-uranium acetate for 5 min on ultrathin sections (80 nm) of prepared retinas, and the ultrastructure of the RPE layer was observed with a transmission electron microscope (x 7000 + 10000).

And (3) observing retinal pigment epithelium by a transmission electron microscope:

as shown in FIGS. 7-9, the cells of the control group of FIG. 7 were normal in morphology, the nuclei were circular or oval, and the boundaries between the cells were clear. In the model group of FIG. 8, the vacuolization of cytoplasm is obvious, the cell membrane is incomplete or broken, and a large number of vacuoles and lipid droplets are visible. FIG. 9ghrelin improves the prognosis of nuclear and cytoplasmic structures, and a large number of vacuoles remain in the cell.