Application of metformin in preparation of medicine for treating cataract

Abstract

The invention belongs to the field of biological medicine, and particularly relates to application of metformin in preparation of a medicine for treating cataract. The invention provides application of metformin in preparation of a medicine for preventing and / or treating cataract. Meanwhile, the invention provides a preparation for preventing and / or treating cataract, and the preparation contains metformin. It is found that metformin has a protective effect on lens epithelial cells, oxidative damage and apoptosis of the lens epithelial cells in an oxidative stress environment can be effectively resisted, and the key effect of SIRT1 / FOXO1 signal channel mediated autophagy in metformin is defined. A clear cell molecular mechanism is provided for treating age-related cataract by using metformin, and the metformin is expected to become a safe and effective medicine for treating cataract.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to the application of metformin in the preparation of drugs for treating cataracts. Background Art

[0002] Cataract refers to a degenerative change in which the optical quality decreases due to a decrease in the transparency or a change in the color of the lens. The main symptoms are progressive and painless vision loss. Patients may also experience symptoms such as decreased contrast sensitivity, glare, monocular diplopia or polyopia, color vision changes, and visual field defects. The lens is an important part of the eye's refractive system and is the only refractive medium with the ability to adjust. The lens consists of a lens capsule, lens epithelium, lens fibers, and zonular fibers. The lens is like the "lens" of the eye and is normally transparent. When it becomes cloudy, it will affect the entry of light into the eye and focus on the retina, resulting in a series of problems such as vision loss. Cataracts occur mostly in people over 40 years old and increase with age. It is related to multiple factors, such as the slow metabolism and degenerative changes in the elderly, and some people also think it is related to long-term sunlight exposure, endocrine disorders, metabolic disorders, etc. Trauma, drugs, radioactive substances, complications, etc. can also cause acquired cataracts. In addition, some congenital cataract patients mostly exist before or after birth, and there are two types: endogenous and exogenous. Endogenous ones are related to fetal developmental disorders, and exogenous ones are caused by systemic diseases of the mother or fetus that damage the lens.

[0003] Cataract is a blinding eye disease that causes vision loss due to lens opacity and is related to factors such as age, ultraviolet rays, and diabetes. At present, surgery is the only effective treatment method. The mainstream surgical methods include phacoemulsification (crushing and aspirating the cloudy lens through a 2-3 mm micro-incision and implanting an intraocular lens, with small trauma and quick recovery) and femtosecond laser-assisted surgery (using a laser to precisely complete steps such as incision making and capsulotomy, improving safety and visual quality). There are various types of intraocular lenses, such as single focus (economical and affordable), multifocal / trifocal (restoring full-range vision), and astigmatism correction type, which need to be selected according to the patient's needs. After the operation, attention should be paid to eye cleaning, avoiding strenuous exercise, and regular reexamination. In recent years, refractive cataract surgery has achieved a technical upgrade from "seeing" to "seeing clearly" through personalized measurement and the application of functional intraocular lenses.

[0004] Age-related cataract (ARC) is the most common cause of visual impairment in middle-aged and elderly people. The prevention and treatment of cataracts have always been the focus of blindness prevention and treatment work in this century. Deeply exploring its pathogenesis and finding effective ways to prevent the occurrence of cataracts or delay their development is not only a global challenge but also a social and economic issue of far-reaching significance. Therefore, finding effective, safe, and inexpensive drugs for the treatment of cataracts has important practical significance. Summary of the Invention

[0005] The purpose of the present invention is to provide the application of metformin in the preparation of drugs for the treatment of cataracts.

[0006] Metformin (1,1-dimethylbiguanide hydrochloride) is a drug widely used in the treatment of diabetes. It is the first-line treatment drug for type 2 diabetes and can be used alone or in combination with other hypoglycemic drugs such as sulfonylureas, glinides, insulin, etc. to better control blood sugar. Currently, there is no report that metformin can be used as a drug for the treatment of cataracts.

[0007] The present invention proposes the hypothesis that "MET can improve the oxidative damage and apoptosis of LECs mediated by regulating the autophagy of the SIRT1 / FOXO1 signaling pathway, thereby delaying the occurrence and development of ARC". By culturing the human lens epithelial cell line HLE-B3 in vitro and inducing its apoptosis with a certain concentration of H2O2 to simulate the occurrence mechanism of ARC, the effects of MET on cell viability, apoptosis, autophagy level, and oxidative stress-related indicators in the oxidative damage model of LECs, as well as the expressions of SIRT1, FOXO1, Ac-FOXO1, etc. were observed to explore the protective mechanism of MET on the oxidative damage of LECs and the role played by autophagy mediated by the SIRT1 / FOXO1 signaling pathway, providing new ideas for further exploring the mechanism of MET protecting LECs and finding drug targets for intervening in the occurrence and development of cataracts.

[0008] Based on the above research, the present invention provides the application of metformin in the preparation of drugs for the prevention and / or treatment of cataracts.

[0009] Preferably, the cataracts include age-related cataracts. Metformin can play a role in preventing and treating various age-related diseases by activating the AMPK-dependent pathway or the non-dependent pathway.

[0010] The present invention provides a preparation for the prevention and / or treatment of cataracts, and the preparation contains metformin.

[0011] Metformin can improve the oxidative damage and apoptosis of LECs mediated by regulating the autophagy of the SIRT1 / FOXO1 signaling pathway, thereby delaying the occurrence and development of ARC.

[0012] The metformin can act on the cell viability, apoptosis, autophagy level and oxidative stress-related indexes of the LECs oxidative damage model, as well as the expressions of SIRT1, FOXO1, Ac-FOXO1, etc.

[0013] After the metformin intervenes in the H2O2-induced oxidative damage of LECs, the apoptosis markers p53, p21, p16 and pro-inflammatory cytokines IL-6, IL-8 in the cells are significantly down-regulated, and the cell survival rate is increased, suggesting that MET can inhibit the apoptosis of LECs under the oxidative stress environment.

[0014] According to the preparation of the specific embodiment of the present invention, the preparation further contains a pharmaceutically acceptable carrier.

[0015] Preferably, the pharmaceutically acceptable carrier is any dispersion medium, coating, isotonic agent compatible with drug administration.

[0016] Preferably, the preparation further contains a pharmaceutically acceptable synergist and / or excipient.

[0017] Preferably, the preparation includes drops, liquids, tablets or drug-loaded sustained-release intraocular lenses.

[0018] Preferably, the preparation is an ordinary preparation, a sustained-release preparation, a controlled-release preparation, a targeted preparation or various particulate drug delivery systems.

[0019] Among the above drugs, the oral preparation includes additives selected from at least one of fillers, diluents, disintegrants, binders, lubricants, glidants, surfactants, solvents, flavoring agents, stabilizers, coloring agents, preservatives.

[0020] The fillers or diluents include saccharides such as lactose, sucrose, glucose, mannitol, sorbitol, dextrin; starches such as starch, pregelatinized starch, dextrin; celluloses such as microcrystalline cellulose, gum arabic, fenugreek gum, dextran; inorganic salts such as calcium sulfate, calcium hydrogen phosphate, pharmaceutical calcium carbonate, light anhydrous silicic acid, synthetic aluminum silicate, calcium silicate, magnesium aluminum silicate.

[0021] The lubricants or glidants or anti-adhesives include stearic acid; metal stearates such as calcium stearate or magnesium stearate; talc powder; colloidal silica; microcrystalline silica gel, hydrogenated vegetable oil; polyethylene glycol, lauryl sulfates such as sodium lauryl sulfate or magnesium lauryl sulfate; silicates such as silicon anhydride or hydrated silicate.

[0022] The described adhesive includes distilled water, ethanol at different concentrations, starch paste, hydroxypropyl methylcellulose, hydroxypropyl cellulose, polyvinylpyrrolidone, methylcellulose, ethylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, polyethylene glycol, and compounds similar to the above excipients.

[0023] The described disintegrant includes cellulose derivatives such as low-substituted hydroxypropyl cellulose, carboxymethyl cellulose, calcium carboxymethyl cellulose, or cross-linked sodium carboxymethyl cellulose; cross-linked polyvinylpyrrolidone; and chemically modified starch / cellulose, such as carboxymethyl starch or sodium carboxymethyl starch.

[0024] The described surfactant includes Tween 80, sodium dodecyl sulfate, sodium stearyl sulfonate, etc.

[0025] The described antioxidant includes sodium bisulfite, sodium metabisulfite, sodium sulfite, anhydrous sodium sulfite, sodium thiosulfate, ascorbic acid, methionine, thiourea, phosphoric acid, citric acid, etc.

[0026] The described preservative or antibacterial agent includes benzoic acid and sodium benzoate, sorbic acid, ethanol, parabens (nipagin esters), benzalkonium bromide, o-phenylphenol, benzyl alcohol, phenethyl alcohol, sodium propionate, sorbic acid, eucalyptus oil, cinnamon oil, and peppermint oil, etc.

[0027] For the usage method of the above drugs, it includes administering an effective amount of the above drugs to a subject. The administration method can be oral, intravenous injection, or transdermal penetration, and applied to the patient in need of treatment.

[0028] Preferably, the administration method of the preparation includes ocular surface administration, intraocular administration, or preparation of a drug-loaded intraocular lens for administration.

[0029] A pharmaceutically effective amount refers to a reasonable benefit / risk ratio that can be obtained by applying the drug for treatment and an amount sufficient to treat the disease. The level of the effective dose can be determined depending on some factors, including the type and severity of the patient's disease, the activity of the drug, the sensitivity to the drug, the administration time, the administration route, the excretion rate, the treatment cycle, the drugs used simultaneously, and other factors well-known in the medical field. The drugs of the present invention can be administered as an independent therapeutic reagent or in combination with other therapeutic reagents. Moreover, the composition of the present invention can continuously or simultaneously add typical therapeutic reagents, and the composition can be administered once or multiple times. It is important to consider all the above factors and administer at the minimum dose that can produce the maximum effect without side effects, and the dose can be determined by a physician according to the patient's condition, age, etc.

[0030] The beneficial effects of the present invention:

[0031] Experimental results show that metformin exhibits significant protective effects in the treatment of cataracts, mainly through multiple mechanisms such as antioxidation, regulation of the redox enzyme system, inhibition of apoptosis, and regulation of the expression of autophagy-related proteins. Specifically, metformin can significantly reduce the accumulation of reactive oxygen species (ROS) induced by oxidative stress, restore the activity of superoxide dismutase (SOD) and decrease the level of malondialdehyde (MDA), thereby alleviating oxidative damage. In addition, metformin can also inhibit oxidative stress-induced apoptosis and regulate the expression of autophagy-related proteins through the SIRT1 / FOXO1 pathway, improve autophagic flux, and protect cells from damage. These results indicate that metformin has significant antioxidant and anti-apoptotic effects, can effectively improve the pathological changes related to cataracts, and is expected to become a safe and effective drug for the treatment of cataracts. Description of the Drawings

[0032] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0033] Figure 1 Showing the oxidative damage effect of TBHP on lens epithelial cells, where

[0034] A shows the results of the effect of different concentrations of TBHP on cell viability;

[0035] B shows the calculation of the IC50 value of TBHP by fitting the curve;

[0036] Figure 2 Showing the effect of metformin on cell viability.

[0037] Figure 3 Showing the protective effect of metformin on BHP-induced oxidative damage.

[0038] Figure 4 Showing the effect of metformin on reactive oxygen species (ROS) in oxidatively damaged cells, where

[0039] A shows the results of detecting intracellular ROS by a fluorescence probe;

[0040] B shows the statistical results of intracellular ROS.

[0041] Figure 5 Showing the effect of metformin on SOD and MDA in oxidatively damaged cells; where

[0042] A shows the effect of metformin on SOD in oxidatively damaged cells;

[0043] B shows the effect of metformin on MDA in oxidatively damaged cells.

[0044] Figure 6 To show the effect of metformin on apoptosis of oxidatively damaged cells, where

[0045] A shows the cell viability by immunofluorescence;

[0046] B shows the statistical results of relative fluorescence density of cells.

[0047] Figure 7 To show the effect of metformin on the expression of related proteins in oxidatively damaged cells;

[0048] A shows the immunofluorescence detection results of related proteins in damaged cells;

[0049] B shows the statistical results of the expression of related proteins. Detailed implementation manners

[0050] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be described in detail below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other implementation manners obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope protected by the present invention.

[0051] Example 1 Establishment of an oxidative stress injury model of lens epithelial cells

[0052] In this example, tert-butyl hydroperoxide (100 μM, 200 μM, 300 μM, 400 μM, 500 μM and 600 μM) was used to treat cells for 6 hours to establish an oxidative stress model, and the cell viability was detected by CCK-8. The specific experimental steps are as follows:

[0053] HLE-B3 cells were seeded in 6-well plates, with about 5×10 5 cells per well, and randomly divided into 5 groups.

[0054] Blank control group (Control group): Human lens epithelial cells HLE-B3 were cultured in DMEM medium.

[0055] Model group (H2O2 group): According to the optimal concentration of H2O2 determined by the previous experimental results, add the optimal concentration of H2O2 to the normally cultured cells and act for 12 h; Low-concentration MET group: Pretreat the cells with 5 μmol.L-1 MET for 24 hours, and then add the medium with the optimal concentration of H2O2 and act for 12 h; Medium-concentration MET group: Pretreat the cells with 15 μmol.L-1 MET for 24 hours, add the medium with the optimal concentration of H2O2, and act for 12 h; High-concentration MET group: Pretreat the cells with 25 μmol.L-1 MET for 24 hours, and then add the medium with the optimal concentration of H2O2 and act for 12 h.

[0056] As Figure 1 shown, with the increase in the concentration of tert-butyl hydroperoxide (TBHP), the cell viability gradually decreases. In the concentration range of 100 - 600 μM, compared with the control group, the cell viability significantly decreases (P < 0.0001). The IC50 value is calculated to be 395.2 μM by fitting the curve, and the cell inhibition rate reaches 50% at this time. Therefore, 400 μM TBHP is selected as the optimal concentration for the subsequent experimental model.

[0057] Example 2 Investigate the effect of metformin on cell viability

[0058] Treat the cells with different concentrations of metformin (0.5 mM, 1.0 mM, 1.5 mM, 2.0 mM, 2.5 mM, 3.0 μM, 3.5 mM, and 4.0 mM) for 24 hours, and detect the cell viability to evaluate its toxicity. The specific experimental steps are as follows:

[0059] Take HLE-B3 cells in good growth state. When they grow to 75% - 80%, discard the original medium, add trypsin for digestion and then resuspend, and perform cell counting. Take a cell suspension with a density of 5×10 5 ml-1 and place 100 μL in each well of a 96-well plate. Set 2 replicates for each group, put it in an incubator at 37 °C with a volume fraction of 5% CO2 for 12 h. When the cells adhere and grow to confluence, add different concentration gradients of H2O2 to each group, which are 33 μmol.L -1 , 50 μmol.L -1 , 100 μmol.L -1 , 200 μmol.L -1 , and act for 0, 3, 12, 24 h respectively. Use a cell counting kit-8 (CCK-8) to detect the cell proliferation rate of each group, and use flow cytometry to detect the apoptosis rate of each group.

[0060] As Figure 2As shown, in the range of 0.5 mM to 2.0 mM, metformin had no significant effect on cell viability, but showed obvious toxicity at 2.5 μM, and the cell viability decreased significantly (P = 0.0081). Therefore, the concentration range of 0.5 μM to 2.0 μM of metformin was selected for subsequent experiments.

[0061] Example 3 investigated the protective effect of metformin on oxidative damage

[0062] After pretreating cells with different concentrations of metformin (0.5 mM, 1.0 mM, 1.5 mM, 2.0 mM) for 24 hours, 400 μM TBHP was used to induce oxidative damage for 6 hours, and cell viability was detected by CCK-8. The specific experimental steps are as follows:

[0063] Take HLE-B3 cells in good growth state. When they grow to 75%-80%, discard the original culture medium, add trypsin to digest and resuspend, and perform cell counting. Seed 1×10 4 HLE-B3 cells into each well of a 96-well plate, set 2 duplicate wells for each group. After they adhere and grow, group and intervene them according to the corresponding experiments, then add 10 μL of CCK-8 solution to each well, incubate for 1.5 h, place it in a full-automatic microplate reader, set the wavelength to 450 nm, and measure the absorbance (A) value of each well.

[0064] Calculate the cell viability according to cell viability = A(treatment group) / A(sample control group) × 100%.

[0065] As Figure 3 shown, metformin treatment could significantly improve the decrease in cell viability induced by TBHP, and 0.5 mM had the most significant effect (P < 0.0001). Therefore, 0.5 mM was selected as the optimal concentration for subsequent experiments.

[0066] Example 4 Effect of metformin on reactive oxygen species (ROS) in oxidatively damaged cells

[0067] The intracellular ROS level was detected using DCFH-DA fluorescent probe. The specific experimental steps are as follows:

[0068] Cell slides were placed in the orifice plate and coated with polylysine. After HLE-B3 cells adhered to the cell slides, they were washed three times with PBS for 5 minutes each time. The cell slides were immersed in 4% paraformaldehyde for fixation at room temperature for 20 minutes, and then washed three times with PBS for 5 minutes each time. Subsequently, they were incubated with SIRT1, Ac-FOXO1, Beclin-1, LC3-II / I, and p62 antibodies respectively. The first incubation was overnight at 4°C. After washing with PBS to remove the residual primary antibody solution, the sections were incubated with fluorescently labeled secondary antibodies at 37°C for 30 minutes; after washing with PBS, DAPI staining solution was incubated at 25°C for 10 minutes to stain the cell nuclei, and then washed with PBS again. Immunofluorescence images were observed and captured under a fluorescence microscope, and the relative immunofluorescence intensity was quantitatively analyzed using Image J software.

[0069] As Figure 4 shown, the intracellular ROS level in the TBHP model group was significantly increased (P<0.0001), and was significantly decreased after metformin pretreatment (P = 0.0005). The results indicated that metformin could effectively reduce the ROS accumulation induced by oxidative stress.

[0070] Example 5 Effects of Metformin on SOD and MDA in Oxidatively Damaged Cells

[0071] The amounts of oxidative stress-related factors SOD, MDA, and MPO in cells of each group were measured using an ELISA kit.

[0072] The standard and diluted sample solutions were successively added to the wells of the ELISA plate, 100 μL per well, and then incubated at 37°C for 1 h; after incubation, the solution was poured out and detection reagents A and B were added to the wells respectively. After each addition of the detection reagent solution, it was incubated at 37°C for 30 min; subsequently, the plate was thoroughly rinsed with PBS-Tween 20 buffer solution and all the residual solution was completely removed. Then the plate was incubated with TMB substrate at 37°C in the dark for 15 min. After that, 50 μL of stop solution was added to each well, and the color changed from yellow to blue to terminate the color reaction. The absorbance at 450 nm was recorded as the absorbance value (A value) using an enzyme-linked immunosorbent assay reader.

[0073] The standard curve and equation were calculated using the A values of the obtained standard solutions. The contents of SOD, MDA, and MPO in cells of each group were determined according to the standard curve and equation.

[0074] As Figure 5As shown, the SOD activity in the TBHP model group decreased significantly, while the MDA level increased significantly (P<0.0001). After pretreatment with metformin, the SOD activity recovered significantly (P = 0.0049), and the MDA level decreased significantly (P = 0.0036). The results indicate that metformin can improve oxidative damage by regulating the redox enzyme system.

[0075] Example 6

[0076] The apoptosis rate of cells was detected by TUNEL, and the specific experimental steps were as follows:

[0077] Take HLE-B3 cells in good growth state. When they grow to 75%-80%, discard the original culture medium, add trypsin for digestion and then resuspend, and perform cell counting. Inoculate HLE-B3 cells into a 6-well plate, adjust the cell concentration to 5×10 7 cells per well. After they adhere and grow, group and intervene them according to the corresponding experiments. Then discard the original culture solution, rinse twice with PBS, add trypsin with a concentration of 2.5 g / L for digestion for 1 min. When it is observed that the cells are floating singly and roundly in the digestive solution, immediately terminate the digestion with the culture medium, centrifuge at 1500 r.min-1 for 10 min, discard the supernatant, resuspend the cells with PBS and perform cell counting again, and adjust to 2×10 5 cells per group, centrifuge at 1000 r.min -1 for 5 min. Slowly add 500 μL of Loading buffer to the centrifuge tube and gently pipette to suspend the cells. Then add 5 μL of AnnexinV FITC and mix well, and then slowly add 5 μL of PI solution. Incubate in the dark at room temperature for 10 min, and then perform flow cytometry detection on the machine.

[0078] As Figure 6 shown, the apoptosis of cells in the TBHP model group increased significantly (P<0.0001), and pretreatment with metformin significantly reduced the apoptosis rate (P = 0.0015). The results indicate that metformin has an inhibitory effect on oxidative stress-induced apoptosis of cells.

[0079] Example 7 investigated the effect of metformin on the expression of related proteins in oxidatively damaged cells

[0080] Add the collected cell samples to the lysis solution for lysis. Homogenize the mixture solution at 12,000 r.min -1, Centrifuge at 4 °C for 10 min. Collect protein samples from the centrifuged supernatant. Subsequently, use a BCA kit to quantify the concentration of the protein samples. Mix 5× Loading Buffer with the protein solution and boil in boiling water for 5 min for sample preparation. After preparing the gel, load the sample, completely separate the proteins by electrophoresis at 80 V, and then transfer them to a 0.22-μm PVDF membrane at a current of 300 mA. Immerse the transferred PVDF membrane in the prepared skim milk blocking solution for 2 h; after blocking, incubate with primary antibodies against SIRT1, FOXO1, Ac-FOXO1, LC3-II / I, Beclin1, and p62 overnight at 4 °C. In this experiment, the GAPDH antibody was used as an internal loading control. The next day, wash the membrane 3 times with TBST buffer (10 min / time); after incubating it with the secondary antibody at room temperature for 2 h. Wash the membrane 3 times with TBST buffer solution (10 min / time). Finally, apply the ECL chemiluminescence working reagent to the washed membrane, observe and capture protein bands using an imaging system, and detect the relative protein expression level using Image J software.

[0081] The results are as Figure 7 shown. In the TBHP model group, the protein expressions of SIRT1 and FOXO1 were significantly decreased (P = 0.0029; P < 0.0001), and the levels of autophagy-related proteins LC3B and SQSTM1 were significantly increased, suggesting that autophagic flux was blocked (P < 0.01). Pretreatment with metformin significantly restored the expressions of SIRT1 and FOXO1 (P = 0.0003; P < 0.0166)) and decreased the levels of LC3B and SQSTM1 (P < 0.01), indicating that autophagic flux was restored. After adding the autophagy inhibitor (3-MA) or the SIRT1 inhibitor (Ex527), the regulatory effect of metformin on protein expression was significantly weakened, further indicating that it improved autophagic flux through the SIRT1 / FOXO1 pathway.

[0082] As described above, it is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention can easily think of changes or substitutions, which should all be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.

Claims

1. Use of metformin in the preparation of drugs for preventing and / or treating cataracts.

2. The use according to claim 1, characterized in that: The cataract includes age-related cataract.

3. A preparation for preventing and / or treating cataract, comprising metformin.

4. The preparation according to claim 3, characterized in that The preparation also contains a pharmaceutically acceptable carrier.

5. The preparation according to claim 4, characterized in that The pharmaceutically acceptable carrier is any dispersion medium, coating, or isotonic agent that is compatible with drug administration.

6. The preparation according to claim 4, characterized in that The preparation may also contain pharmaceutically acceptable enhancers and / or excipients.

7. The preparation according to claim 4, characterized in that The preparation includes drops, liquids, tablets or drug-loaded sustained-release intraocular lenses.

8. The preparation according to claim 3, characterized in that The administration method of the preparation includes ocular surface administration, intraocular administration or administration by preparing drug-loaded intraocular lens.

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