CN114099678A

CN114099678A - Lens damage inhibitor based on Keap1-Nrf2 signal channel

Info

Publication number: CN114099678A
Application number: CN202111394361.5A
Authority: CN
Inventors: 白洁; 杨帆
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2021-11-23
Filing date: 2021-11-23
Publication date: 2022-03-01

Abstract

The invention discloses a lens damage inhibitor based on a Keap1-Nrf2 signal channel, which belongs to the field of lens damage inhibition, contains a mitochondrion targeting peptide, can inhibit lens damage, particularly damage of epithelial cells, through the Keap1-Nrf2 signal channel, can improve the internal oxidative stress level and cell activity of LECs, improves the inhibition effect on oxidative stress induced apoptosis, has potential beneficial benefits, has no obvious toxic or side effect, has good clinical application prospect, and provides important theoretical and practical basis for clinical diagnosis and treatment.

Description

Lens damage inhibitor based on Keap1-Nrf2 signal channel

Technical Field

The invention relates to the field of lens damage inhibition, in particular to a lens damage inhibitor based on a Keap1-Nrf2 signal pathway.

Background

The world health organization reports that cataract is the leading blinding eye disease worldwide. The results of an "age-related eye disease study" published by the national institute of ophthalmology (NIH) indicate that: cataract is the leading cause of vision deterioration and blindness, and as the average life of human beings is prolonged, cataract blind people become public health problems which cannot be ignored.

At present, no effective method for preventing cataract from generating and developing exists internationally, cataract surgery is the first choice method for treating cataract, but surgery still has certain risks, obvious economic and psychological burdens are brought to patients, huge expenditure is increased to society, and factors such as expensive surgical equipment and materials limit the cataract development of people in less-developed areas particularly in remote mountainous areas and less-developed areas of China. Therefore, a new economic and effective way for preventing and treating cataract without operation is explored, is an important measure for coping with the aging of the population in China, and has potential application prospect and wide social benefit.

Disclosure of Invention

In view of the problems in the prior art, the invention aims to provide a lens damage inhibitor based on a Keap1-Nrf2 signal pathway.

In order to solve the above problems, the present invention adopts the following technical solutions.

A lens injury inhibitor based on a Keap1-Nrf2 signaling pathway, the inhibitor comprising a mitochondrial targeting peptide that inhibits lens injury through a Keap1-Nrf2 signaling pathway.

Preferably, the lens damage comprises lens epithelial cell damage.

The lens damage inhibitor is applied to the preparation of a preparation for inhibiting ROS expression in human lens epithelial cells.

The lens damage inhibitor is applied to preparation of the inhibitor for inhibiting oxidative damage and apoptosis of human lens epithelial cells.

The verification method for inhibiting the lens damage by the inhibitor based on the Keap1-Nrf2 signal path comprises the following steps:

s1: grouping of cells

Culturing LECs in a culture solution, and grouping cell lines when the cell fusion reaches 85-95%; filtering SS-31 with microporous filter membrane, and storing diluted substances in serum-free culture medium at low temperature; SS-31 control group: treating the cells with SS-31 with different amount concentrations of the diluted substances respectively; SS-31 Experimental group: treating the cells with SS-31 at the concentrations of the different substances after dilution, and then with H₂O₂Processing; preferably, the culture solution is a DMEM culture solution containing 10% heat-inactivated fetal calf serum; preferably said H₂O₂The mass concentration of the substance is 300 mu mol/L;

s2: cell viability assay

Analyzing the effect of SS-31 on the survival rate of LECs by CCK-8; the CCK-8 analysis process includes culturing LECs in 96-well plate at cell density of 1 × 10⁵one/mL, then 10 μ L of CCK-8 solution was added per well and incubated at 37 ℃ for 1 hour, and absorbance at 450nm was detected using a microplate reader;

s3: flow cytometry for detecting apoptosis rate

Collecting cells, suspending the cells in a BindingBuffer, adding PI and annexinV-FITC solution, incubating at room temperature in a dark place, and performing fluorescence detection by using a flow cytometer; preferably, the annexin V-FITC maximum excitation light is 488nm, and the emission light is 520 nm; the maximum excitation light of PI is 535nm, and the emission light is 617 nm;

s4: intracellular ROS level determination

Inoculating cells and culturing overnight; after the cell density reaches 80-90%, performing dyeing treatment; then observing by using a microscope, wherein the microscope can be a fluorescence microscope or a laser confocal microscope; the specific process of the dyeing treatment is to prepare H by DMSO₂DCFDA, remove the culture medium of the cultured cells until no residue, add 1mL1 XH per well₂Incubating DCFDA working solution in a cell culture box at 37 ℃ for 30 minutes;

s5: western-blot detection of keap1 protein and Nrf2 protein expression in LECs

Cell lysis with RIPA lysis buffer, 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis separation of equivalent amount of protein, and transfer to polyvinylidene fluoride membrane, after skimmed milk powder closed, adding primary antibody at 2-6 deg.C overnight, incubating the membrane with goat anti-rabbit IgG secondary antibody combined with horseradish peroxidase at room temperature, washing the membrane for multiple times, adding ECL luminescence solution, and analyzing the relative expression amount of protein with ImageJ software. Preferably, the dilution concentration of the goat anti-rabbit IgG secondary antibody combined with horseradish peroxidase is 1: 2000.

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

the inhibitor provided by the scheme inhibits lens damage based on a Keap1-Nrf2 signal channel, contains mitochondrial targeting peptide, and can improve the oxidative stress level and cell activity in LECs, so that the inhibition effect on oxidative stress-induced cell apoptosis can be improved, potential beneficial benefits are provided, no obvious toxic or side effect is generated, the clinical application prospect is good, and important theoretical and practical basis is provided for clinical diagnosis and treatment.

Drawings

FIG. 1 is a bar graph of the cell viability of example 1 of the present invention after addition of the present inhibitor; FIG. 2 is a bar graph of cell viability in example 1 of the present invention without the addition of inhibitors;

FIG. 3 shows administration of H to a control group in example 1 of the present invention₂O₂+ 0.1. mu.M SS-31 cell morphology observation scheme; FIG. 4 shows administration of H to a control group in example 1 of the present invention₂O₂+ 1. mu.M SS-31 cell morphology visualization scheme; FIG. 5 shows administration of H to a control group in example 1 of the present invention₂O₂+ 10. mu.M SS-31 cell morphology visualization scheme;

FIG. 6 shows the administration of H to the experimental group of example 1 of the present invention₂O₂+ 0.1. mu.M SS-31 cell morphology observation scheme; FIG. 7 shows the administration of H to the experimental group of example 1 of the present invention₂O₂+ 1. mu.M SS-31 cell morphology visualization scheme; FIG. 8 shows the administration of H to the experimental group of example 1 of the present invention₂O₂+ 10. mu.M SS-31 cell morphology visualization scheme;

FIG. 9 shows administration of H to a control group in example 1 of the present invention₂O₂+ 0.1. mu.M SS-31 flow cytometry apoptosis detection scheme; FIG. 10 shows administration of H to a control group in example 1 of the present invention₂O₂+ 1. mu.M SS-31 flow cytometry apoptosis detection scheme; FIG. 11 shows administration of H to a control group in example 1 of the present invention₂O₂+ 10. mu.M SS-31 flow cytometry apoptosis detection scheme;

FIG. 12 shows the administration of H to the experimental group of example 1 of the present invention₂O₂+ 0.1. mu.M SS-31 flow cytometry apoptosis detection scheme; FIG. 13 shows the administration of H to the experimental group of example 1 of the present invention₂O₂+ 1. mu.M SS-31 flow cytometry apoptosis detection scheme; FIG. 14 shows the administration of H to the experimental group of example 1 of the present invention₂O₂+10 μ M SS-31 flow cytometric apoptosis assayA schematic diagram;

FIG. 15 is a statistical bar graph of the proportion of apoptotic cells in example 1 of the present invention;

FIG. 16 shows administration of H to a control group in example 1 of the present invention₂O₂+ 0.1. mu.M after SS-31H₂The DCFDA staining results show a schematic; FIG. 17 shows administration of H to a control group in example 1 of the present invention₂O₂+ 1. mu.M after SS-31H₂The DCFDA staining results show a schematic; FIG. 18 shows administration of H to a control group in example 1 of the present invention₂O₂+ 10. mu.M after SS-31H₂The DCFDA staining results show a schematic;

FIG. 19 shows the administration of H to the experimental group of example 1 of the present invention₂O₂+ 0.1. mu.M after SS-31H₂The DCFDA staining results show a schematic; FIG. 20 shows the administration of H to the experimental group of example 1 of the present invention₂O₂+ 1. mu.M after SS-31H₂The DCFDA staining results show a schematic; FIG. 21 shows the administration of H to the experimental group of example 1 of the present invention₂O₂+ 10. mu.M after SS-31H₂The DCFDA staining results show a schematic;

FIG. 22 shows process H in example 1 of the present invention₂O₂Schematic representation of keap1 protein expression in induced LECs; FIG. 23 shows scheme H in example 1 of the present invention₂O₂Induced Nrf2 protein expression profiles within LECs; FIG. 24 shows process H in example 1 of the present invention₂O₂Schematic representation of GAPDH protein expression within induced LECs;

FIG. 25 is a Western-blot detection Keap1-Nrf2 pathway protein expression histogram in example 1 of the present invention.

Detailed Description

Example 1: verification method for inhibiting lens damage based on Keap1-Nrf2 signal channel

S1: grouping of cells

LECs cells were cultured in DMEM medium containing 10% heat-inactivated fetal bovine serum. Cell lines were grouped when cell fusion reached around 90%. SS-31 was filtered through a microporous membrane (0.22 μm) and diluted to 0.1. mu. mol/L, 1. mu. mol/L and 10. mu. mol/L in serum-free medium, and stored in a 4 ℃ refrigerator for further use. SS-31 control group: respectively using SS-31 (0.1. mu. mol/L, 1. mu. mol/L and 10. mu. mol)L) treating the cells for 24 h; SS-31 Experimental group: after treatment of the cells with SS-31 (0.1. mu. mol/L, 1. mu. mol/L and 10. mu. mol/L) for 24h, 300. mu. mol/L H was added₂O₂And treating for 24 h. Further experiments were performed 48 hours after induction of each group of cells.

S2: cell viability assay

The effect of SS-31 on cell viability of LECs was analyzed by CCK-8. LECs cells were cultured in 96-well plates (cell density 1X 10)⁵one/mL), 10 μ L of CCK-8 solution was added per well, and incubated at 37 ℃ for 1 hour, and absorbance at 450nm was measured using a microplate reader.

S3: flow cytometry for detecting apoptosis rate

Collecting cells, and mixing 1X 10⁵Each cell was resuspended in 200. mu.L of BindingBuffer, 4. mu.L of 0.5mg/mL PI and 2. mu.L of annexin V-FITC solution were added, incubated for 15min at room temperature in the dark, and subjected to fluorescence detection using a flow cytometer. (AnnexinV-FITC maximum excitation 488nm, emission 520 nm. pi maximum excitation 535nm, emission 617nm, however, both can be excited by 488nm laser.)

S4: intracellular ROS level determination

The appropriate number of cells were inoculated and cultured overnight at 37 ℃. And (4) dyeing after the cell density reaches 80-90%. DMSO preparation 10mM H₂DCFDA, remove the culture medium of the cultured cells until no residue, add 1mL1 XH per well₂DCFDA working solution is incubated in a cell culture box at 37 ℃ for 30 minutes and observed by a fluorescence microscope or a laser confocal microscope.

S5: western-blot detection of keap1 protein and Nrf2 protein expression in LECs

The cell is lysed by RIPA lysis buffer, equivalent protein is separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (80V30min, 120V90min), the cell is transferred to a polyvinylidene fluoride (PVDF) membrane, the cell is sealed by 5% skimmed milk powder for 1h, a primary antibody (diluted concentration 1:1000) is added, the cell is kept overnight at 4 ℃, the membrane is incubated for 1h by a goat anti-rabbit IgG secondary antibody (diluted concentration 1:2000) combined by Horse Radish Peroxidase (HRP) at room temperature, ECL luminescence solution is added after the membrane is washed for 3 times, and the relative expression amount of the protein is analyzed by ImageJ software.

Analytical example 1:

as shown in FIGS. 1-2, the amount and concentration of the inhibitor and H added to the culture medium are different₂O₂Stem prognosis, histogram of survival of human lens epithelial cells; as can be seen from the figure, the cell survival rate was not significantly affected after SS-31 intervention in LECs, and 300. mu. M H was administered₂O₂The survival rate of LECs is obviously reduced, while SS-31 can inhibit H₂O₂The survival rate of the induced LECs decreased.

As shown in FIGS. 3-8, the LECs in the control group and 1. mu.M SS-31 group were morphologically regular, with clear intercellular spaces of 300. mu. M H₂O₂In the stem prognosis, the volume of LECs is obviously reduced, the cell spacing is increased, part of cells are changed in a spherical shape, wiredrawing change occurs among cells, and SS-31 stem prognosis and the morphology of LECs are gradually improved.

As shown in fig. 9-14, each panel has four quadrants, the lower left quadrant representing the proportion of normal viable cells, the upper left quadrant representing the proportion of mechanically damaged cells, the upper right quadrant representing the proportion of apoptotic cells, and the lower right quadrant representing the proportion of necrotic cells; FIG. 15 is a histogram of statistics analyzing the third quadrant cell proportion, i.e., the proportion of apoptotic cells; by flow cytometry, it was shown that₂O₂The induced apoptosis rate of LECs is obviously increased, while SS-31 can inhibit H₂O₂Induced apoptosis of LECs with decreasing apoptosis rate with increased SS-31 dose;

as shown in FIGS. 16-21, for the detection of intracellular ROS levels, ROS-sensitive probe H was used₂DCFDA. DCFH-DA (2',7' -dichlorofluorescein diacetate) is a non-labeled oxidation-sensitive fluorescent probe, does not have fluorescence per se, can freely pass through a cell membrane, can be hydrolyzed by intracellular esterase to generate DCFH after entering the cell, and DCFH can not permeate the cell membrane, so that the probe can be easily loaded into the cell, active oxygen in the cell can oxidize the non-fluorescent DCFH to generate fluorescent DCF, and the level of the active oxygen in the cell can be known by detecting the fluorescence of the DCF. H₂DCFDA staining results showed that the LECs in the control group and 1. mu.M SS-31 group both exhibited uniformly low fluorescence, H₂O₂Induced appearance of brilliant green in LECsThe color is high in fluorescence, which represents that the ROS level in cells is increased, after SS-31 stem prognosis with different doses, the intensity of bright green high fluorescence in LECs is gradually reduced, and the SS-31 can effectively inhibit the ROS expression in the LECs.

As shown in FIGS. 22-25, H₂O₂The expression of the keap1 protein in the induced LECs is obviously increased, and the SS-31 can inhibit H₂O₂An increase in the expression of the induced keap1 protein; h₂O₂Induced reduction of Nrf2 protein expression in LECs, while SS-31 was able to promote H₂O₂The induced increase of the Nrf2 protein expression proves that the Keap1-Nrf2 signal path is in SS-31 to H₂O₂Plays an important role in the process of induced LECs injury.

Claims

1. A lens damage inhibitor based on a Keap1-Nrf2 signal pathway, characterized in that: the inhibitor contains a mitochondrially targeted peptide that inhibits lens damage through the Keap1-Nrf2 signaling pathway.

2. The lens damage inhibitor based on the Keap1-Nrf2 signaling pathway of claim 1, wherein: the lens damage includes lens epithelial cell damage.

3. Use of an inhibitor according to claim 1 for the manufacture of a formulation for inhibiting the expression of ROS in human lens epithelial cells.

4. The use of the inhibitor according to claim 1 for the preparation of a medicament for inhibiting oxidative damage and apoptosis of human lens epithelial cells.

5. The lens damage inhibitor based on the Keap1-Nrf2 signaling pathway of claim 1, wherein: the verification method for inhibiting the lens damage by the inhibitor based on the Keap1-Nrf2 signal pathway comprises the following steps:

s1: grouping of cells

Culturing LECs in culture medium until cell fusion reaches 85-95%At that time, the cell lines are grouped; filtering SS-31 with microporous filter membrane, diluting with serum-free culture medium to different material amount and concentration, and storing at low temperature; SS-31 control group: treating the cells with SS-31 with different amount concentrations of the diluted substances respectively; SS-31 Experimental group: treating the cells with SS-31 at the concentrations of the different substances after dilution, and then with H₂O₂Processing;

s2: cell viability assay

Analyzing the effect of SS-31 on the survival rate of LECs by CCK-8;

s3: flow cytometry for detecting apoptosis rate

Collecting cells, suspending the cells in a BindingBuffer, adding PI and annexinV-FITC solution, incubating at room temperature in a dark place, and performing fluorescence detection by using a flow cytometer;

s4: intracellular ROS level determination

Inoculating cells and culturing overnight; after the cell density reaches 80-90%, performing dyeing treatment; then observing by a microscope;

s5: western-blot detection of keap1 protein and Nrf2 protein expression in LECs

Cell lysis with RIPA lysis buffer, 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis separation of equivalent amount of protein, and transfer to polyvinylidene fluoride membrane, 5% skimmed milk powder sealing, adding primary antibody 2-6 deg.C overnight, incubating the membrane with goat anti-rabbit IgG secondary antibody combined with horseradish peroxidase at room temperature, washing the membrane for several times, adding ECL luminescence solution, and analyzing the relative expression amount of protein with ImageJ software.

6. The lens damage inhibitor based on the Keap1-Nrf2 signaling pathway of claim 5, wherein: in S1, the culture solution is a DMEM culture solution containing 10% heat-inactivated fetal bovine serum; said H₂O₂The concentration of the substance was 300. mu. mol/L.

7. The lens damage inhibitor based on the Keap1-Nrf2 signaling pathway of claim 5, wherein: s2, the CCK-8 analysis comprises culturing LECs in 96Cell density in the well plate is 1X 10⁵one/mL, then 10. mu.L of CCK-8 solution per well was added and incubated at 37 ℃ for 1 hour, and the absorbance at 450nm was measured using a microplate reader.

8. The lens damage inhibitor based on the Keap1-Nrf2 signaling pathway of claim 5, wherein: in S3, the maximum excitation light of the annexin V-FITC is 488nm, and the emission light is 520 nm; the PI maximum excitation light was 535nm and the emission light was 617 nm.

9. The lens damage inhibitor based on the Keap1-Nrf2 signaling pathway of claim 5, wherein: s4, the specific process of the dyeing treatment is to prepare H by DMSO₂DCFDA, remove the culture medium of the cultured cells until no residue, add 1mL1 XH per well₂DCFDA working solution, incubated in a cell culture chamber at 37 ℃ for 30 minutes.

10. The lens damage inhibitor based on the Keap1-Nrf2 signaling pathway of claim 5, wherein: in S5, the dilution concentration of the horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody is 1: 2000.