阅读说明：本技术 一种天然分子pa在预防、治疗细胞或组织损伤制剂中的应用 (Application of natural molecule PA in preparation for preventing and treating cell or tissue injury ) 是由 傅瑶 孙浩 周梦 张春雷 于 2021-10-19 设计创作，主要内容包括：本发明提供一种天然分子植酸,Phytic acid,简称PA在治疗细胞或组织损伤制剂中的应用,利用天然分子PA的强螯合能力,将其应用于治疗铁死亡或氧化应激损伤的细胞或组织的药物中,抑制细胞内芬顿反应和羟基自由基的产生,维持氧化应激反应的平衡,达到抗铁死亡,预防、治疗细胞或组织损伤的目的。(The invention provides application of natural molecule Phytic Acid (PA) in a preparation for treating cell or tissue injury, which utilizes the strong chelating capacity of the natural molecule PA to apply the natural molecule PA to a medicament for treating cells or tissues with iron death or oxidative stress injury, inhibits the generation of Fenton reaction and hydroxyl free radicals in cells, maintains the balance of oxidative stress reaction, and achieves the purposes of resisting iron death and preventing and treating cell or tissue injury.)

1. The application of a natural molecule PA in a preparation for preventing and treating cell or tissue damage, wherein the structural formula of the natural molecule PA is as follows:

2. the use according to claim 1, wherein the cell or tissue damage is caused by iron death or an imbalance in oxidative stress.

3. Use according to claim 2, wherein the iron death is iron death induced by UV light.

4. Use according to claim 1, wherein the cells or tissue are corneal endothelial cells or corneal endothelium.

5. The use according to claim 4, further comprising one or more of the following features:

1) the preparation is a medicament for inhibiting lipid compound peroxidation;

2) the preparation is a medicament for preventing or treating cell mitochondrial damage;

3) the preparation is a medicament for preventing or treating the reduction of ZO-1 expression in cells;

4) the preparation is a medicament for preventing or treating cell nucleus damage.

6. The use according to any one of claims 1 to 5, wherein the formulation is a liquid formulation.

7. The use according to claim 1, wherein the agent is a medicament for preventing or treating oxidative stress injury diseases.

8. The use according to claim 7, wherein the oxidative stress injury disease comprises Parkinson's disease, Alzheimer's disease, tumors, stroke, ischemia-reperfusion injury.

Technical Field

The invention relates to the technical field of anti-iron death and oxidative stress drugs, in particular to application of a natural molecule PA in a preparation for preventing and treating cell or tissue injury.

Background

Phytic Acid (PA) commonly known as inositol hexaphosphate and having a molecular formula of C₆H₁₈O₂₄P₆Molecular weight of 660.04, widely found in beans, grains, and nuts. Each phytic acid molecule contains 6 phosphate groups, can chelate with iron ions, copper ions and the like in a wider pH range to form a stable complex, and has a large number of applications in the fields of metal corrosion prevention, rust prevention and the like. The phytic acid is used as a natural nontoxic compound and has a huge application prospect in the field of biomedicine.

Iron death (Ferroptosis) is an iron-dependent, novel programmed cell death modality distinguished from apoptosis, necrosis, and autophagy. The main features of iron death include peroxidation of cell membrane lipids, elevated levels of cellular Reactive Oxygen Species (ROS), reduced mitochondrial shrinkage, and the like. The classical iron death inhibitor Ferrostatin-1(Fer-1) is an artificially synthesized antioxidant which inhibits cell death by preventing membrane lipid peroxidation through a reduction mechanism. The expensive synthesis cost of Fer-1 is limited, and the application of Fer-1 in the field of biomedicine is limited due to poor water solubility, so that the development of natural organic molecules with low cost, safety, no toxicity and excellent performance as iron death inhibitors has important scientific value and application prospect. The application of the natural molecule PA in the anti-iron death and oxidative stress medicines is not reported.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, the present invention aims to provide a natural molecule PA for use in a preparation for preventing and treating cell or tissue damage.

In order to achieve the above objects and other related objects, the present invention provides a use of PA, a natural molecule, in a preparation for preventing and treating cell or tissue damage.

The application of a natural molecule PA in a preparation for preventing and treating cell or tissue damage, wherein the structural formula of the natural molecule PA is as follows:

preferably, the cell or tissue damage is caused by iron death or an imbalance in oxidative stress.

Preferably, the iron death is iron death induced by ultraviolet light.

Preferably, the cell or tissue is a corneal endothelial cell or a corneal endothelium.

Preferably, the agent is a drug that inhibits peroxidation of a lipid compound.

Preferably, the agent is a medicament for treating mitochondrial damage in cells.

Preferably, the agent is an agent for preventing or treating a decrease in the expression of ZO-1 in cells;

preferably, the agent is a medicament for preventing or treating nuclear damage of cells.

Preferably, the formulation is a liquid formulation.

As a further preference, the formulation is a liquid formulation for direct treatment of iron death injury or oxidative stress injury.

As a further preference, the formulation is an eye drop for treating iron death injury or oxidative stress injury of corneal endothelium.

As a further preference, the formulation may be used for the treatment of oxidative stress injury diseases, such as parkinson's disease, alzheimer's disease, tumors, stroke, ischemia-reperfusion injury, and the like.

The invention aims to use the natural molecule PA to maintain the balance of oxidative stress reaction by chelating ferrous ions so as to achieve the aim of anti-iron death, thereby being used for preventing and treating the injury of tissues and organs and the like, and verifying the feasibility of the application of the natural molecule PA in the medical field through experiments.

As described above, the use of the natural molecule PA of the present invention in a preparation for preventing and treating cell or tissue damage has the following beneficial effects:

1. the invention utilizes the natural molecule PA with good antioxidant activity, and the natural molecule PA can be applied to the medicines for treating cells or tissues with iron death or oxidative stress damage, can effectively chelate ferrous ions, inhibit the fenton reaction and the generation of hydroxyl free radicals in cells, maintain the balance of oxidative stress reaction, and achieve the purposes of resisting iron death and preventing and/or treating the cell or tissue damage.

2. Experiments prove that the natural molecule PA plays a role in protecting the cell morphology of ultraviolet-induced corneal endothelial injury cells, can inhibit the generation and accumulation of lipid peroxides in the ultraviolet-induced corneal endothelial injury cells, can promote the growth and proliferation of the ultraviolet-induced corneal endothelial injury cells, promotes the ZO-1 expression of the ultraviolet-induced corneal endothelial injury cells and protects the size and the morphology of cell mitochondria, and has good prospects in the aspect of treating oxidative stress injury diseases such as Parkinson's disease, Alzheimer's disease, tumors, stroke, ischemia-reperfusion injury and the like through other approaches.

Drawings

FIG. 1 is a diagram of cell morphology before and after UV-induced corneal endothelial injury.

FIG. 2 is a graph showing fluorescence detection of intracellular lipid peroxides after UV-induced corneal endothelial injury.

FIG. 3 is a statistical chart of the fluorescence detection results of intracellular lipid peroxides after UV-induced corneal endothelial injury.

FIG. 4 is a fluorescent flow cytometric assay of intracellular lipid peroxides after UV-induced corneal endothelial injury.

FIG. 5 is a statistical chart of the fluorescent flow cytometry detection of intracellular lipid peroxides after UV-induced corneal endothelial injury.

FIG. 6 is a graph showing the growth of UV-induced corneal endothelial injury cells.

FIG. 7 is a graph showing the expression of ZO-1 in UV-induced corneal endothelial injury cells.

FIG. 8 is a graph showing mitochondrial changes in UV-induced corneal endothelial injury cells.

FIG. 9 is a diagram showing the cell contour and the cell nucleus structure change in the process of ultraviolet ray induced endothelial cell injury of rabbit corneal tissue.

FIG. 10 is a diagram showing the changes of cell contour and cell nucleus structure during the process of damaging human corneal tissue endothelial cells by ultraviolet rays.

FIG. 11 is a cell location diagram of Nrf2 protein during the process of ultraviolet ray induced endothelial cell injury of rabbit corneal tissue.

FIG. 12 is a statistical chart of the accumulation of Nrf2 protein in cell nucleus during the process of ultraviolet ray-induced endothelial cell injury of rabbit corneal tissue.

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

The corneal endothelial cells are the innermost layer of the five-layer structure of the cornea, have no regeneration capacity after in vivo injury, and have the similar structure and the most representative structure with most cells. UV-A accounts for 95% of UV in the environment and can cause damage to the cornea located on the surface layer.

The following experiments were all performed on corneal endothelial cells. The culture medium used for each group was as follows:

control group: corneal endothelial culture fluid;

fer-1 group: a cornea endothelium culture solution with Fer-1 concentration of 5 mu mol/L;

PA group: a corneal endothelial culture solution with a PA concentration of 1 mu mol/L;

UV group: corneal endothelial culture fluid;

UV + Fer-1 group: a cornea endothelium culture solution with Fer-1 concentration of 5 mu mol/L;

UV + PA group: the concentration of PA in the corneal endothelial culture solution was 1. mu. mol/L.

Example 1: isolation and culture of rabbit corneal endothelial cells

The cells are taken from a New Zealand white rabbit, the rabbit is euthanized, the rabbit is placed in a supine position, the upper eyelid is clamped by using high-pressure sterilized toothed forceps, one assistant turns the upper eyelid outwards, an operator clamps conjunctiva tissues by using the toothed forceps with one hand and shears from the conjunctival epithelium and stroma with the other hand, the upper eyelid is separated towards two sides along the eyelid margin, a bulbar conjunctiva part is clamped outwards from the limbus part of an eyeball, the bulbar conjunctiva is separated by the shears along the sphere, the conjunctiva tissues at two sides are clamped after complete separation, and the upper eyelid is cut short along the fornix. After the conjunctiva is separated, one hand holds the forceps to clamp the rest conjunctival tissue, and the other hand holds the elbow to shear the tissues around the eyeball and the optic nerve, so that the eyeball is completely separated. The eyeball was completely immersed for 30s and washed twice with PBS.

Another clean sterile 10cm petri dish was laid with a piece of sterile gauze, a hand held forceps was used to clamp an eyeball and placed on the gauze, another hand held sterile blade was used to pierce the ball from the back side of the corneosclera margin, the scissors were changed, the cut-in portion was entered through the puncture, the cut-out corneosclera margin was cut open, the cut-out corneosclera was placed in OPTI solution containing 1% double antibody (penicillin 10 KU/mL-streptomycin 10mg/mL mixed solution) in serum-free, and placed on ice. Prepare the stereomicroscope, gooseneck, adjust the stereomicroscope until the cornea is visible intact. Holding the tooth microscopic forceps on the left hand, holding the toothless microscope on the right hand, closing the forceps head by the right hand, drawing several times from the sclera to the cornea until the single-layer endothelial cells roll up, and changing the direction until the whole corneal endothelial layer is separated (note that if the endothelial cells can not be completely separated, the peripheral endothelial tissues are separated as far as possible, and the peripheral endothelial cells have stronger proliferation capacity and better activity than the middle endothelial cells).

The intact detached corneal endothelium was subjected to 0.1% collagenase digestion for 14-16 h. On the next day, the completely digested corneal endothelial cells were blown several times with a 1mL pipette (1000. mu.L, ependorff), observed under a microscope until they were dispersed into a uniform small cell mass, digested by adding a serum-containing culture solution, centrifuged at 8000rpm for 4min, and the supernatant was removed. Adding 1ml of tryptle (pancreatin) and beating several times, placing at 37 deg.C and 5% CO₂Incubator digestion 7And taking out every 3min and blowing for 10min until the small cell mass is separated into single cells.

Spreading Fibronectin in 6-well plate, culturing overnight in incubator, and collecting Fibronectin in 6-well plate in super clean bench for air drying. The corneal endothelial cells thus obtained were all seeded in one well of a 6-well plate, and 2mL of corneal endothelial culture medium (45mLopti +4mLFBS + 0.5. mu.LEGF + 500. mu.l diabody + 2. mu.LSB 431542+ 50. mu.LY 27632) was added and cultured. And observing the cell state by Day2 (the next Day) and Day3 (the third Day), photographing under a microscope, and changing the liquid every 48 hours until the cell density reaches 80%, so that the cell can be passaged at a passage ratio of 1: 3.

Example 2: PA plays a role in protecting the cell morphology of ultraviolet-induced corneal endothelial injury

The experimental method comprises the following steps: the culture medium in the dish was aspirated, washed twice with PBS (phosphate buffered solution, PH 7.4), and the cell morphology was observed under an optical microscope and photographed. The washed cells were divided into six groups, i.e., a control group, a Fer-1 group, a PA group, a UV + Fer-1 group, and a UV + PA group.

Control group: adding corneal endothelial culture solution, culturing for 6h, and observing cell morphology under optical microscope;

fer-1 group: adding a corneal endothelial culture solution with Fer-1 concentration of 5 mu mol/L for culturing for 6h, and observing the morphology of cells under an optical microscope;

PA group: adding a corneal endothelial culture solution with PA concentration of 1 mu mol/L for culturing for 6h, and observing the morphology of cells under an optical microscope;

UV group: adding corneal endothelium culture solution, placing into UV ultraviolet accelerated aging test box, and irradiating at a power of 1.75J/cm²Carrying out ultraviolet UV-A irradiation for 240s, washing twice by PBS after irradiation is finished, observing the cell morphology under an optical microscope, and taking a picture; then adding corneal endothelial culture solution to culture for 6h, and observing the cell morphology every 2h under an optical microscope;

UV + Fer-1 group: adding the corneal endothelium culture solution with Fer-1 concentration of 5 μmol/L, placing into UV ultraviolet accelerated aging test box, and irradiating at power of 1.75J/cm²Ultraviolet UV-A irradiation is carried out for 240s, and PBS is used for washing after the irradiation is finishedTwice, observing the cell morphology under an optical microscope, and taking a picture; then adding a corneal endothelial culture solution containing Fer-1 to culture for 6h, and observing the morphology of the cells under an optical microscope every 2 h;

UV + PA group: adding corneal endothelium culture solution with PA concentration of 1 μmol/L, placing into UV ultraviolet accelerated aging test box, and irradiating at power of 1.75J/cm²Carrying out ultraviolet UV-A irradiation for 240s, washing twice by PBS after irradiation is finished, observing the cell morphology under an optical microscope, and taking a picture; then, a corneal endothelial culture solution containing PA was added to the cells to culture the cells for 6 hours, and the morphology of the cells was observed under an optical microscope every 2 hours.

And (4) analyzing results: as shown in fig. 1, all cells maintained the same cell density and status prior to UV-a treatment, eliminating the effect of initial status on the experiment. The growth and proliferation rates of cells without any treatment (Control group, shown as Control) were in a normal state, and cells cultured in the Fer-1 medium (corneal endothelial medium containing Fer-1) (Fer-1 group) exhibited similar behavior to the Control group, indicating that Fer-1 was not cytotoxic to CEC, and cells cultured in the PA medium (corneal endothelial medium containing PA) (PA group) exhibited similar behavior to the Control group, indicating that PA was not cytotoxic to CEC. While in the UV group, 1.75J/cm is accepted²Abnormal cell morphology appeared in the cells after UV-A irradiation, and the number of cells was significantly reduced after 6 h; the number and the shape of the cells treated by the Fer-1 culture medium are superior to those of the UV group; however, the PA-treated cells (UV + PA group) remained proliferating and appeared in a regular shape, indicating that PA protected the UV-A-treated cells and that the protection was more pronounced after 6h of action.

Example 3: PA can inhibit ultraviolet-induced corneal endothelial injury cell from producing lipid peroxide

The experimental method comprises the following steps: the experiment uses a lipid ROS detection kit, cell plating is carried out according to groups, when the density is close to 80%, the cell is pretreated by using corresponding culture solution for 12 hours, PBS is used for cleaning twice, ultraviolet irradiation is carried out to induce damage, PBS is added for cleaning twice after irradiation is finished, the cell morphology is observed under an optical microscope, a picture is taken, culture solution containing BODIPY 581/591 is added for culture, the cell is observed once under a fluorescence microscope every 3 hours, and a fluorescence image is recorded.

Control group: adding corneal endothelial culture solution containing BODIPY 581/591, culturing for 12h, observing cell morphology under a fluorescence microscope, and taking a fluorescence photograph;

UV group: (cell-free culture solution, in dry state) was put into a UV ultraviolet accelerated aging test chamber, and the irradiation power was 1.75J/cm²Irradiating for 240s with ultraviolet UV-A, washing twice with PBS after irradiation, adding corneal endothelial culture solution containing BODIPY 581/591, culturing for 12h, observing cell morphology under a fluorescence microscope, and taking a fluorescence photograph;

UV + PA group: (cell-free culture solution, in dry state) was put into a UV ultraviolet accelerated aging test chamber, and the irradiation power was 1.75J/cm²Irradiating for 240s with ultraviolet UV-A, washing twice with PBS after irradiation, adding a corneal endothelial culture solution containing BODIPY 581/591 with PA concentration of 1 μmol/L, culturing for 12h, observing cell morphology under a fluorescence microscope, and taking a fluorescence photograph;

and (4) analyzing results: the production and accumulation of Lipid peroxides (Lipid ROS) is the largest feature of iron death. BODIPY 581/591 undecanoic acid is used for detecting the presence of ROS (reactive oxygen species) as a lipid in cells and membranes, and once an oxidation reaction occurs, the structure of BODIPY 581/591 undecanoic acid is changed, so that the fluorescence emission peak is changed from-590 nm to-510 nm, and a change from red to green is observed under a fluorescence microscope. The change in lipid peroxide following UV-a irradiation of corneal endothelial cells was detected using BODIPY 581/591 undecanoic acid. As can be seen from fig. 2, the cells of the control group (without any treatment) emitted only red fluorescence, and almost no green fluorescence was expressed; in contrast, in the UV-a treated group (subjected to UV-a radiation), most cells fluoresce green, which means that many cells produce lipid ROS upon UV-a stimulation, leading to oxidation of BODIPY 581/591 undecanoic acid, and the green fluorescence expression of corneal endothelial cells treated with PA is greatly reduced compared to cells irradiated with single UV-a, reversing the immunofluorescent staining change of lipid ROS reaction. FIG. 3 is a statistical result based on the fluorescence intensity of FIG. 2, and the result is consistent therewith.

Example 4: fluorescent flow cytometry detection of lipid peroxides

The experimental method comprises the following steps: the time point at which fluorescence was most pronounced in example 3 was recorded, the cells at that time were digested, and flow cytometric analysis was performed. Cells marked as PI fluorescence were counted for the number of valid cells, and cells marked as FITC fluorescence were cells with lipid peroxide production.

And (4) analyzing results: digesting the cells with fluorescence reaction, performing flow detection, and performing statistics by using mean value in the result, as shown in fig. 4, compared with the cell peak value of the control group, the peak value after UV treatment is shifted to the right, which means that the cells with the target fluorescence (i.e. green fluorescence) are increased; the peak of the cells treated with PA was left falling back, i.e., the cells with green fluorescence became fewer. Statistical analysis of FIG. 4 resulted in the flow-through statistic of FIG. 5, in which UV-A stimulated more cells to produce the lipid ROS, while PA decreased by the ratio consistent with the results of the fluorescence experiment.

Example 5: PA can promote ultraviolet ray induced corneal endothelial injury cell growth and proliferation

The experimental method comprises the following steps: cell plating was performed according to the grouping, and CCK8 experiments were performed using 96-well plates. And (4) Day0, adding corresponding culture solution containing 10% CCK8 solution into 1000 cells per well according to groups, standing for 4 hours in an incubator, measuring the absorbance at 450nm by using an enzyme-labeling instrument, and storing data. Day1, completely sucking culture solution, placing a cell culture plate to be irradiated in a sterilized ultraviolet irradiation box for irradiation, washing with PBS for three times, adding corresponding culture solution containing 10% CCK8 solution according to groups, standing for 4 hours in the culture box, measuring absorbance at 450nm by an enzyme-labeling instrument, and storing data. Day3, sucking out the culture solution, washing with PBS for three times, adding corresponding culture solution containing 10% CCK8 solution according to groups, standing for 4h in an incubator, measuring absorbance at 450nm by using an enzyme-labeling instrument, and storing data. All data were normalized to Day0 and plotted statistically.

And (4) analyzing results: data from three experiments were collected and normalized to day0 data to generate statistical data, as shown in fig. 6, with similar growth curves for the control group (black, no treatment group) and the PA group (dark gray, using the PA-containing medium group), and with a statistical result of NS, no significant statistical difference, indicating that PA had no negative effect on cell growth on day0, day1, and day 3; in both groups receiving UV-A, day1 data showed a marked decrease in cell viability for both UV (silver) groups without PA protection and UV + PA (light grey) groups with PA; on day3, however, the UV + PA (light grey) group containing PA showed a tendency to increase cell proliferation and number, while the UV (silver) group without PA protection showed a further decrease in cell number. It can be seen that PA not only does not negatively affect cell proliferation without any treatment, but also promotes better recovery of cells to normal state in the case of UV-a damage.

Example 6: PA can promote the expression of ZO-1 in the corneal endothelial injury cells induced by ultraviolet rays

The experimental method comprises the following steps: the P1 generation cells in good state are seeded in a 24-well plate paved with cell slide according to groups, when the density is close to 80%, the cells are pretreated for 12h by using corresponding culture solution, and the cells are washed twice by PBS. UV irradiation induced damage was performed, fixation was performed at Beform, 0h, 6h time points of irradiation, i.e. 4% paraformaldehyde fixed at room temperature for 15min, and PBS washing was performed three times. The donkey serum blocking solution is used for blocking for 1h at a warm temperature, PBS is used for washing for three times, ZO-1 primary antibody prepared by the blocking solution is used for incubating overnight in a cold storage at 4 ℃, PBST + Tween is used for washing for three times, and the last time is used for cleaning. The murine fluorescent secondary antibody, formulated with PBS, was incubated for 1h at room temperature and washed three times with PBST + Tween. Preparing a clean glass slide, dripping DAPI on the glass slide, carefully clamping a cell slide, slowly placing the side with cells on the glass slide, standing for 5min, and observing and shooting under a fluorescence microscope in a dark condition.

And (4) analyzing results: complete expression of ZO-1 reflects the intact structure of CEC, as shown in FIG. 7, three groups of cells all showed a complete green outline by ZO-1 immunofluorescence staining prior to UV-A irradiation, blue representing the nucleus; in cells without any treatment (control group), the number of cells and the expression of tight junctions slightly increased with time; in the UV-A group, the expression of ZO-1 is reduced at 0h of irradiation, and after 6h, not only the expression of ZO-1 is reduced, but also the number of cell nuclei represented by blue is greatly reduced; while the UV + PA group had no significant protective effect at the instant time point of 0h, the corneal endothelial cell tight junction expression was increased and the number of nuclei was restored to normal level at the same time point of 6 h.

Example 7: PA can protect mitochondria of ultraviolet-induced corneal endothelial injury cells

The experimental method comprises the following steps: the culture solution in the suction culture dish, add PBS and wash twice, observe the cell morphology under the optical microscope, open the UV ultraviolet ray aging test case with higher speed, adjust time is 240s, put into the laboratory case with the cell culture dish that washs and carry out ultraviolet irradiation, add PBS after the illumination is accomplished and wash twice, observe the cell morphology again under the optical microscope, add culture solution and cultivate 6h, digest the cell and get off the back and compete with the stationary liquid heavy suspension with the electricity, hand over the electron microscope and shoot in the laboratory.

And (4) analyzing results: as shown in fig. 8, the normal control group had normal cell mitochondria size and intact morphology; mitochondrial volume becomes smaller, number of cristae is reduced, and membrane shrinkage occurs after ultraviolet UV-A injury; after Fer-1 is used, the mitochondrial volume of the cells is recovered, the number of ridges is slightly increased, and the shrinkage of membranes is reduced; the mitochondrial volume of the cells after the PA is used is basically recovered to be normal, the number of ridges is increased, and the membrane shrinkage is reduced, which shows that the mitochondrial protection effect and the mitochondrial damage treatment effect of the PA on the UV-induced corneal endothelial damaged cells are superior to that of Fer-1.

Example 8: PA can protect the structure and the nuclear integrity of damaged endothelial cells on ultraviolet-induced corneal tissue and promote Nrf2 to gather in the nuclear

In vitro organ culture experimental method: taking a fresh rabbit eyeball, cutting the rabbit eyeball along the edge of the corneosclera, completely stripping the iris tissue by using a toothed forceps, putting the cornea tissue into a 24-hole plate along the inner surface upwards, and adding culture solution of a corresponding group for pretreatment for 12 hours. Washing with PBS twice, irradiating with ultraviolet ray to induce injury, washing with PBS twice after irradiation, and culturing in culture solution for 6 hr. Adding 5% donkey serum blocking solution into each group of corneal holes, sealing for 1h at a constant temperature, washing with PBS for three times until no donkey serum blocking solution remains, incubating in a refrigerator at 4 ℃ for overnight with ZO-1 primary antibody (1:200), washing with PBS for three times, incubating with murine secondary antibody at room temperature for 1h, and washing with PBS for three times. Under a stereomicroscope, the endothelial layer was peeled off as completely as possible using an ophthalmological microscope, the endothelial cells were placed face down on a glass slide on which DAPI was dropped, and the cover slip was carefully covered to expel air bubbles as far as possible. In the dark, the images were observed and photographed under a fluorescence microscope.

And (4) analyzing results: as shown in FIG. 9, in the case of no UV-A treatment, the staining of three endothelial cell layers all showed complete cell contour, wrapping the normal cell nucleus structure, the control group without any treatment showed the normal cell structure at the time points of 0h and 6h as well as the before, while in the instant of UV-A irradiation, the integrity of the cell membrane was destroyed and the cell nucleus was also affected, some of the cell nucleus became small, the regular oval shape was lost, and after 6h, not only the expression of ZO-1 was greatly reduced, but also the normal cell nucleus was almost absent. In sharp contrast, the PA-treated cell layer preserved a partial membrane structure immediately upon UV-a irradiation and restored the tight junctions of most cells after 6h, forming a more complete cell structure. In addition to the organ culture experiments of rabbit tissue, human corneal tissue was used to validate the results. The cornea scleral ring after the corneal transplantation is adopted in the experiment, similar to rabbit cornea treatment, after other tissues such as iris and the like are stripped, the cornea scleral ring is divided into three groups, corresponding culture medium is used for pretreatment for 12 hours, then UV-A radiation treatment is carried out, cells are fixed by paraformaldehyde after culture is continuously carried out for 6 hours by using the culture medium, an endothelial ring of the corneal scleral edge is separated under a stereoscopic microscope, ZO-1 is used for staining, and DAPI represents cell nucleus. As shown in FIG. 10, before UV-A irradiation, all groups of cells showed hexagonal cobblestone contour outlined by tight junction protein, the shape of nucleus was elliptical but smaller than that of rabbit endothelial cells, and the green border did not completely cover the nucleus, which resulted from narrow scleral ring, damaged separation process, difficulty in complete separation, resulting in CEC slices overlapping and not being completely laid on the same horizontal plane.

The change of the cellular localization of the Nrf2 protein is observed in the process of damaging corneal endothelial cells by UV-A through in vitro organ culture experiments, as shown in figure 11, complete blue cell nuclei can be observed in three groups before UV-A treatment, Nrf2 (red) is uniformly stained and distributed around the nuclei, and the part of red and blue which are overlapped is almost not existed, so that the distribution of Nrf2 is in cytoplasm and accords with the distribution condition under the normal condition. At the same time of 6h, the Control group cells maintain the original state, the UV group cells have no expression of Nrf2, the cell nucleus is damaged, although the red and blue overlap is increased, so that a part of Nrf2 enters the cell nucleus, and the UV treated by PA irradiates the group cells, the cell nucleus is intact, and a large amount of red expression exists in the cell nucleus part, so that more Nrf2 is gathered in the cell nucleus and corresponds to the protein distribution of the functional state. As can be seen from fig. 12, in the PA-treated 6H group, the nuclear Nrf2 ratio was larger, showing that more Nrf2 was concentrated in the nucleus.

From the above experimental results, it can be known that the natural molecule PA of the present invention has a protective effect on the cell morphology of the uv-induced corneal endothelial injury cell, can inhibit the generation and accumulation of lipid peroxides in the uv-induced corneal endothelial injury cell, can promote the growth and proliferation of the uv-induced corneal endothelial injury cell, and can promote the expression of the uv-induced corneal endothelial injury cell ZO-1 and protect the size and morphology of the cell mitochondria. The in vitro culture of rabbit and human corneal tissues proves that the natural molecule PA can protect the ultraviolet ray induced corneal endothelial damaged cell structure and complete cell nucleus and promote Nrf2 to be gathered in the cell nucleus, so that the natural molecule PA preparation can be used for preparing eye drops to directly treat the corneal endothelium damaged by UV or oxidative stress, and has good prospects in treating oxidative stress damage diseases such as Parkinson's disease, Alzheimer's disease, tumors, stroke, ischemia-reperfusion injury and the like through other ways.

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.