Application of metformin

Abstract

The invention relates to the field of biological medicine, in particular to application of metformin. The research finds that the metformin regulates and controls an AMPK / B-RAF / ERK signal channel, and further regulates and controls an important signal molecule SMAD2 / 3 in the epithelial-mesenchymal transition process. The invention not only provides a research basis for the development of targets of crystalline lens epithelial cell epithelial-mesenchymal transition related pathway signal molecules, but also provides a scientific basis for the development of new candidate drugs for preventing and treating posterior capsular opacification, and has important significance for the development of new drugs.

Description

[0001] This application claims the priority of the Chinese patent application filed with the China Patent Office on June 12, 2024, with application number 202410752615.3 and invention name “Application of Metformin”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present invention relates to the field of biomedicine technology, and in particular to the application of metformin. Background Art

[0003] Cataract is a common disease among the elderly and the main cause of blindness, affecting more than 53 million people worldwide. Cataract surgery is the primary treatment method in the world today, but it is often accompanied by some complications, among which posterior capsular opacification (PCO) is one of the most common complications. This complication refers to abnormal proliferation, migration and differentiation of lens epithelial cells remaining in the capsule after being stimulated by cytokines, chemokines and growth factors, resulting in obstruction of light propagation in the visual axis and affecting visual quality.

[0004] Under normal circumstances, lens epithelial cells (LECs) are spread under the anterior capsule and the equator of the lens. Their function is to maintain the transport of electrolytes and fluids in the lens. Studies have shown that the dysfunction of LECs remaining in the lens capsule is considered to be one of the causes of PCO. Therefore, the main method for studying the PCO model is to culture LECs in vitro. According to the differences in pathological changes of PCO, it can be divided into fibrotic PCO and proliferative PCO. The first pathological change is fibrotic change, which mainly refers to the high proliferation of lens epithelial cells, cell transdifferentiation into spindle morphology, matrix deposition and posterior capsule shrinkage. However, during cataract surgery, the blood-water barrier may be destroyed, thereby releasing transforming growth factor β (TGF-β), interleukins, interferons and other cytokines. The cytokines released in this process are bound to aggravate the excessive abnormal proliferation and differentiation of residual cells. Among them, TGF-β2 is the main isomer in the aqueous humor. It activates the Smad pathway and the non-Smad pathway, activates the phosphorylation of the carboxyl terminus of the intracellular signal effector protein Smad (mainly Smad2 and Smad3), and then activates Smad2 or Smad3, which then combines with Smad4 to form a tripartite and translocates to the cell nucleus, forming a heteromeric complex with the TGF-β ligand, thereby regulating the typical TGF-β signaling pathway, activating downstream fibrosis reactions, promoting the occurrence of EMT, and further inducing PCO. Currently, TGF-β2 is the cytokine used in most studies of PCO models.

[0005] Studies have found that the incidence of posterior capsule opacity in cataract patients 2-5 years after surgery is 20-40%. In view of the increasing incidence of posterior capsule opacity after cataract surgery, no drug has been proven to be truly effective in clinical trials at home and abroad so far. Nd:YAG laser posterior capsulotomy is currently a general method for treating posterior capsule opacity. Although it plays a positive role, this surgery brings a certain mental and economic burden to patients. In addition, the main complications of Nd:YAG laser posterior capsulotomy are: increased intraocular pressure, corneal epithelial damage, iris hemorrhage, intraocular lens damage, vitreous anterior boundary rupture, macular cystoid edema and retinal detachment. Therefore, the discovery of effective drugs is a hot topic in the basic research of posterior capsule opacity, which has far-reaching scientific significance, social significance and clinical application prospects. Summary of the invention

[0006] In view of this, the present invention provides the use of metformin.

[0007] Application of metformin in the preparation of medicines for treating complications after cataract surgery.

[0008] In some embodiments, the post-cataract surgery complications include posterior capsule opacification, abnormal proliferation of residual lens epithelial cells after surgery, lens epithelial cell hyperplasia caused by migration, posterior lens capsule opacification and / or decreased visual acuity after cataract surgery.

[0009] In some specific embodiments, the complication after cataract surgery is posterior capsule opacification.

[0010] In some embodiments, the treatment includes: inhibiting epithelial-mesenchymal transition of human lens epithelial cells, inducing autophagy and / or apoptosis of lens epithelial cells, inducing G0 / G1 phase arrest of lens epithelial cells, regulating the expression of EMT-related markers, and regulating the expression of EMT-related pathway proteins.

[0011] In some specific embodiments, the inhibiting epithelial-mesenchymal transition of human lens epithelial cells comprises: inhibiting cell viability, migration and / or proliferation of lens epithelial cells;

[0012] The inducing lens epithelial cell autophagy comprises: increasing the expression level of LC3Ⅱ / Ⅰ protein and / or decreasing the expression level of p62 protein;

[0013] The regulating the expression of EMT-related markers includes: reducing the expression levels of α-SMA and Fibronectin and / or increasing the expression level of the epithelial marker E-Cadherin;

[0014] The EMT-related pathways include at least one of (1) to (3):

[0015] (1) reducing the expression ratio of at least one of the following proteins: p-ERK / ERK, pB-RAF / B-RAF, p-SMAD / SMAD;

[0016] (2) increase the ratio of p-AMPK / AMPK protein expression;

[0017] (3) Increase the expression levels of p-AMPK and AMPK proteins.

[0018] In some embodiments, the effective concentration of metformin is 2.5-40 mM, preferably 5-20 mM, specifically 5 mM, 10 mM or 20 mM.

[0019] In some embodiments, the administration of metformin includes: ocular surface administration, intraocular administration, or administration in the preparation of drug-loaded intraocular lenses.

[0020] The present invention also provides a medicine for preventing and treating cataract complications, comprising metformin and pharmaceutically acceptable adjuvants.

[0021] In some embodiments, the drug for preventing and treating cataract complications is specifically a drug for preventing and treating capsule opacity, abnormal proliferation of residual lens epithelial cells after surgery, lens epithelial cell hyperplasia caused by migration, posterior lens capsule opacity and / or decreased vision after cataract surgery. In some specific embodiments, the drug for preventing and treating cataract complications is specifically a drug for preventing and treating capsule opacity.

[0022] In some embodiments, the dosage form of the drug includes drops, liquids, drops, liquids, tablets, or drug-loaded sustained-release intraocular lenses.

[0023] The present invention also provides a method for preventing and treating cataract complications, comprising: administering metformin or administering the drug of the present invention.

[0024] In the method of the present invention, the metformin or the drug is administered via ocular surface administration, intraocular administration or administration by preparing a drug-loaded intraocular lens.

[0025] The present invention found that metformin regulates SMAD2 / 3, an important signaling molecule in the epithelial-mesenchymal transition process, by regulating the AMPK / B-RAF / ERK signaling pathway. This not only provides a research basis for the development of targets for signaling molecules in the epithelial-mesenchymal transition-related pathways of lens epithelial cells, but also provides a scientific basis for the development of new candidate drugs for the prevention and treatment of posterior capsule opacification, which is of great significance for the development of new drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Shows the effect of metformin pretreatment on (SRA01 / 04) cell viability;

[0027] Figure 2 The effect of metformin pretreatment on the proliferation ability of (SRA01 / 04) cells; 2A: EDU staining immunofluorescence images of cells in each group (EDU×400, scale = 50μm) 2B: Comparison of the percentage of EDU positive cells in each group Compared with the NC group, a P < 0.05 (one-way ANOVA, Tukey test);

[0028] Figure 3 Comparison of cell migration in each group; A: Cell scratch test images of each group at 0h and 18h ​​(×100, scale bar = 200μm) B: Comparison of cell migration rate in each group, * P < 0.05 (one-way ANOVA, Tukey test, n = 3);

[0029] Figure 4 The expression of α-SMA, Fibronectin and E-Cadherin proteins in each group of cells is shown; A: Electrophoresis of α-SMA, Fibronectin and E-Cadherin protein expression B: Comparison of relative expression of Fibronectin protein C: Comparison of relative expression of E-Cadherin protein D: Comparison of relative expression of α-SMA protein, * P < 0.05 (one-way ANOVA, Tukey test, n = 3) 1: NC group; 2: metformin-treated group; 3: TGF-β2-treated group; 4: TGF-β2+metformin group;

[0030] Figure 5 Figure 2 Fluorescence staining method was used to detect the expression of α-SMA and Fibronectin proteins; A: The expression of α-SMA and Fibronectin proteins in different treatment groups (×400, scale bar = 50 μm) B: Comparison of fluorescence intensity of α-SMA in different groups C: Comparison of fluorescence intensity of Fibronectin in different groups, * P < 0.05 (one-way ANOVA, Tukey test, n = 3) α-SMA and Fibronectin were labeled with α-SMA and Fibronectin antibodies, respectively (red), and cell nuclei were labeled with DAPI (blue);

[0031] Figure 6The expression of SMAD2 / 3, AMPK, ERK and B-RAF proteins in each group of cells is shown; A: electrophoresis of SMAD2 / 3, AMPK and ERK protein expression B: comparison of relative expression of p-AMPK / AMPK protein C: comparison of relative expression of p-SMAD2 / 3 / SMAD2 / 3 protein D: comparison of relative expression of p-ERK / ERK protein E: comparison of relative expression of pB-RAF / B-RAF protein, * P < 0.05 (one-way ANOVA, Tukey test, n = 3) 1: NC group; 2: metformin-treated group; 3: TGF-β2-treated group; 4: TGF-β2+metformin group; where “p-” represents phosphorylation;

[0032] Figure 7 The expression of P62 and LC3II / LC3I proteins in each group of cells is shown; A: electrophoresis of P62 and LC3II / LC3I protein expression B: comparison of relative expression of P62 protein C: comparison of relative expression of LC3II / LC3I protein, * P < 0.05 (one-way ANOVA, Tukey test, n = 3) 1: NC group; 2: metformin-treated group; 3: TGF-β2-treated group; 4: TGF-β2+metformin group;

[0033] Figure 8 Fluorescence detection of LC3Ⅱ / Ⅰ protein and p62 expression;

[0034] Fig. 9 Shows the cell cycle distribution in the NC group and the Metformin-treated group;

[0035] Fig.10 Shows the cell apoptosis in NC group and Metformin-treated group;

[0036] Fig.11 The results show the effect of metformin eye drops on the expression level of α-SMA. DETAILED DESCRIPTION

[0037] The present invention provides the application of metformin. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0038] The present invention has the following beneficial effects:

[0039] 1) TGF-β2 induced EMT model in lens epithelial cells in vitro and analyzed the relationship between TGF-β2-induced EMT and cell autophagy;

[0040] 2) Human lens epithelial cells were treated with TGF-β2 or TGF-β2 and metformin in combination, and the regulatory effect of metformin on TGF-β2-induced EMT was further analyzed by immunoblotting analysis of EMT markers, immunofluorescence staining detection of EMT markers, cell scratch assay and morphological observation.

[0041] 3) Human lens epithelial cells were treated with TGF-β2 or TGF-β2 and metformin combined, and the SMAD2 / 3, ERK1 / 2 signaling pathways and AMPK-B-RAF signaling pathways were analyzed by immunoblotting to further analyze the effect of metformin on the TGF-β2-induced signaling pathways;

[0042] 4) It was first discovered that metformin has an inhibitory effect on TGF-β2-induced epithelial-mesenchymal transition of human lens epithelial cells, and it shows good time and concentration dependence;

[0043] 5) Metformin may inhibit B-RAF phosphorylation by activating AMPK, and the inhibited B-RAF regulates ERK phosphorylation, thereby affecting the SMAD signaling pathway. Its mechanism of action was explored.

[0044] 6) Metformin eye drops can inhibit the expression of EMT markers in the in vivo puncture model.

[0045] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0046] The present invention will be further described below in conjunction with embodiments:

[0047] Example 1

[0048] 1. Cell Culture

[0049] The cryovial of frozen human lens epithelial cells (SRA01 / 04) was taken out from the liquid nitrogen tank, immediately placed in a 37°C water bath for rapid thawing, centrifuged, the supernatant was removed, and a culture medium containing 10% fetal bovine serum and antibiotics (100U / mL penicillin and 100mg / mL streptomycin) was added. After mixing by pipetting, the culture bottle was transferred to a culture bottle and placed in an incubator at 37°C and containing 5% CO2 by volume for 48 hours for subsequent experimental detection. Cells in the logarithmic growth phase were used for experiments.

[0050] 2. Cell Viability Assay

[0051] Cell viability was assessed using CCK-8. Cells were seeded in 96-well plates (4 × 10 3 / well), and after overnight culture, cells were treated with concentration gradient (2.5, 5, 10, 20, 40 mM) of Met for 24 h, 48 h, and 72 h, respectively. Subsequently, cells were incubated with 10 μl CCK-8 at 37 ° C for another hour. The absorbance (A) value at 450 nm was measured on an ELISA reader. Cell proliferation rate = (absorption value of the experimental group - absorption value of the blank control) / (absorption value of the control group - absorption value of the blank control) × 100%.

[0052] The results of the CCK-8 experiment showed that cell viability decreased with the increase of metformin concentration, showing a dose-dependent downward trend. In addition, when metformin was used to treat cells at different time points, cell viability decreased in a time-dependent manner ( Figure 1 ).

[0053] 3. Cell proliferation assay

[0054] Cell proliferation was assessed using the EdU imaging kit. Preheated EdU staining solution with a final concentration of 10 μM was added to each group of cells and cultured in a cell culture incubator at 37°C and 5% CO2 for 2 h. After incubation, fix the cells with 4% paraformaldehyde solution at room temperature for 15 min; remove the 4% paraformaldehyde and soak the slides in PBS for 5 min × 2 times. Prepare Click-iT reaction solution (volume per well of a 24-well plate: Clock Reaction Buffer 107.5 μl; CuSO4 5 μl; Azide 555 0.25 μl; ClickAdditive Solutin 12.5 μl) and incubate at room temperature in the dark for 30 min. Remove the Click reaction solution and soak the slides in PBS for 5 min × 2 times. Add DAPI until the cells are completely covered to counterstain the nuclei. Remove the DAPI, soak the slides in PBS for 5 min × 2 times, and take pictures using a fluorescence microscope.

[0055] The results of the EDU experiment showed that the cell proliferation ability decreased with the increase of metformin concentration. The EDU positive cell rates in the 5.0mmol / LMetformin group, 10mmol / LMetformin group and 20mmol / L Metformin group were significantly decreased compared with the NC group, and the differences were statistically significant (P<0.05, all P<0.01) ( Figure 2 B).

[0056] In the subsequent experiments, the metformin concentration in the metformin treatment group and the metformin+TGF-β2 treatment group was 5 mM, and the treatment time of each cell was 24 h.

[0057] 4. Cell scratch assay

[0058] SRA01 / 04 cells were cultured normally, and when the cells grew well, the cells were treated according to the following groups and collected for scratch test. The specific groups were as follows: 1. NC group: cells were cultured normally for 48 hours; 2B. metformin treatment group: cells were treated with 5mM metformin for 24 hours, and then cells were cultured normally for 24 hours; 3. TGF-β2 treatment group: cells were cultured normally for 24 hours, and then cells were treated with 10ng / ml TGF-β2 for 24 hours; 4. metformin+TGF-β2 treatment group: cells were treated with 5mM metformin for 24 hours, and then cells were treated with 10ng / ml TGF-β2 for 24 hours. Then, cell scratch test was performed, and cells in each group were cultured until the density was confluent. Before the experiment, the culture medium was replaced with serum-free culture medium and 1μg / ml mitomycin C was added for 1 hour. Each group of cells was scratched using a 200μl pipette tip, and the cell surface was washed once with serum-free medium to remove cell debris. The cells were observed under a microscope (100×) and photographed by group. The position of the cells in the photographs was recorded for subsequent photography, and serum-free medium was used for culture. Each group of cells was placed in an incubator at 37°C and 5% CO2 for 0h and 18h, and photographed and recorded. The scratch area was measured using ImageJ software, and the scratch healing rate = (scratch area 0h after scratching - scratch area 48h after scratching) / scratch area 0h after scratching × 100%.

[0059] The results of the cell scratch test showed that the cell migration rate in the TGF-β2-treated group was significantly higher than that in the NC group and the TGF-β2+metformin-treated group (all P<0.001). There was no significant difference in the cell migration rate between the NC group and the TGF-β2+metformin-treated group (P>0.05). Figure 3 B)

[0060] 5. Western blotting to detect the expression of EMT-related markers in cells

[0061] After the cells were in good growth condition, the cells were treated according to the following groups: 1. NC group: cells were cultured normally for 48 hours; 2. metformin treatment group: cells were treated with 5mM metformin for 24 hours, and then cells were cultured normally for 24 hours; 3. TGF-β2 treatment group: cells were first cultured normally for 24 hours, and then cells were treated with 10ng / ml TGF-β2 for 24 hours; 4. metformin+TGF-β2 treatment group: cells were treated with 5mM metformin for 24 hours, and then cells were treated with 10ng / ml TGF-β2 for 24 hours.

[0062] The cells were lysed with RIPA lysis buffer containing protease inhibitors, and the total protein was extracted using a total protein extraction kit. The protein concentration was determined using the BCA method. The proteins were separated using SDS-PAGE gel and transferred to a PVDF membrane. The membrane was blocked with 5% skim milk powder at room temperature for 1 hour, and the primary antibody was added for incubation at 4°C overnight. The primary antibody was diluted with TBST. The next day, the membrane was washed, and then the horseradish peroxidase-conjugated secondary antibody was reacted slowly on a shaker at room temperature for 60 minutes. Finally, the membrane was quickly washed 3 to 8 times with TBST solution on a shaker for 5 minutes each time. The ECL luminescent solution was prepared in a 1:1 ratio and dropped onto the membrane to observe the protein expression under the imager. The results are shown in the figure. Figure 4 .

[0063] The results showed that compared with the NC group, the expression levels of α-SMA and Fibronectin in the TGF-β2 treatment group were significantly increased, and the expression level of E-Cadherin was significantly decreased, and the differences were statistically significant (all P<0.001). However, compared with the TGF-β2 treatment group, the fluorescence intensity of SMA and Fibronectin in the TGF-β2+metformin treatment group was significantly decreased (P<0.001, P<0.01), and the expression level of E-Cadherin was significantly increased (P<0.05).

[0064] 6. Immunofluorescence staining to detect EMT-related markers in cells

[0065] After the cells were in good growth condition, the cells were treated according to the following groups: 1. NC group: cells were cultured normally for 48 hours; 2. metformin treatment group: cells were treated with 5mM metformin for 24 hours, and then cells were cultured normally for 24 hours; 3. TGF-β2 treatment group: cells were first cultured normally for 24 hours, and then cells were treated with 10ng / ml TGF-β2 for 24 hours; 4. metformin+TGF-β2 treatment group: cells were treated with 5mM metformin for 24 hours, and then cells were treated with 10ng / ml TGF-β2 for 24 hours.

[0066] The cultured cell slides of each group were fixed in 4% paraformaldehyde for 15 minutes, and the 4% paraformaldehyde was removed and rinsed with PBS 3 times, each time for 5 minutes. 0.1% tritonX-100 was added until the cells were completely covered and incubated at room temperature for 30 minutes, and 0.1% tritonX-100 was removed and rinsed with PBS 3 times, each time for 5 minutes. 1% BSA was added until the cells were completely covered and incubated at room temperature for 15 minutes. α-SMA and Firbronectin primary antibodies were diluted 1:100 with PBS, added until the cells were completely covered, and incubated overnight at 4°C. The primary antibodies were removed and rinsed with PBS for 5 minutes × 3 times. Cy3-labeled goat anti-rabbit IgG secondary antibodies were added at a dilution ratio of 1:200, added until the cells were completely covered, and incubated at room temperature for 60 minutes. Rinse in PBS for 5 minutes × 3 times. DAPI was added until the cells were completely covered to counterstain the nuclei, and then the slides were rinsed with PBS 3 times, each time for 5 minutes. Finally, add anti-fluorescence quenching agent to the slide, put the slide upside down on the slide with anti-fluorescence quenching agent, and observe the staining effect under a fluorescence microscope. Figure 5 .

[0067] The results of immunofluorescence staining showed that the fluorescence intensity of α-SMA and Fibronectin in the TGF-β2 treatment group was significantly increased compared with that in the NC group, and the difference was statistically significant (all P<0.001). However, the fluorescence intensity of SMA and Fibronectin in the TGF-β2+metformin treatment group was significantly decreased compared with that in the TGF-β2 treatment group, and the difference was statistically significant (P<0.001, P<0.01).

[0068] 7. Western blotting method to detect the expression of EMT-related pathway proteins in cells

[0069] When the cells are growing well, treat the cells in the following groups:

[0070] Group 1 (NC group): cells were cultured normally for 48 h;

[0071] Group 2 (metformin-treated group): cells were treated with 5 mM metformin for 24 h, and then cultured normally for 24 h;

[0072] Group 3 (TGF-β2-treated group): cells were first cultured normally for 24 h, and then treated with 10 ng / ml TGF-β2 for 24 h;

[0073] Group 4 (metformin+TGF-β2 treatment group): cells were treated with 5 mM metformin for 24 h, and then treated with 10 ng / ml TGF-β2 for 24 h.

[0074] The cells in each group treated as above were lysed with RIPA lysis buffer containing protease inhibitors, and the total protein was extracted using a whole protein extraction kit. The protein concentration was determined using the BCA method. The protein was separated using SDS-PAGE gel and transferred to a PVDF membrane. The membrane was blocked with 5% skim milk powder at room temperature for 1 hour, and the primary antibody was added for incubation at 4°C overnight. The primary antibody was diluted with TBST. The next day, the membrane was washed, and then the horseradish peroxidase-conjugated secondary antibody was reacted slowly on a shaker at room temperature for 60 minutes. Finally, the TBST solution was used to quickly wash 3 to 8 times on a shaker, each time for 5 minutes. The ECL luminescent solution was prepared in a 1:1 ratio and dropped onto the membrane to observe the protein expression under the imager.

[0075] The results showed that the relative expression ratios of p-ERK / ERK and pB-RAF / B-RAF proteins in the TGF-β2 treatment group were significantly increased compared with those in the NC group, and the difference was statistically significant (all P<0.001); the relative expression ratios of proteins in the TGF-β2+metformin treatment group were significantly decreased compared with those in the TGF-β2 treatment group, and the difference was statistically significant (all P<0.001). In addition, the relative expression ratios of p-AMPK and AMPK proteins in the metformin treatment group were significantly increased compared with those in the NC group and the TGF-β2 treatment group, and the difference was statistically significant (all P<0.01); the relative expression ratios of proteins in the TGF-β2+metformin treatment group were significantly increased compared with those in the TGF-β2 treatment group, and the difference was statistically significant (P<0.05)( Figure 6 ).

[0076] 8. Detection of cell autophagy markers by Western blotting

[0077] The experiment was performed according to the grouping and experimental methods in the experiment of detecting the expression of EMT-related pathway proteins in cells by Western blotting method, and the protein expression levels of LC3Ⅱ / Ⅰ protein and p62 were detected. The results are shown in Figure 7 .

[0078] The results showed that metformin could increase the protein expression level of LC3Ⅱ / Ⅰ in SRA01 / 04 cells treated with TGF-β2, while reducing the protein expression level of p62, suggesting that metformin could further enhance the autophagic flux of TGF-β2-treated LECs.

[0079] 9. Immunofluorescence staining to detect autophagy-related markers in cells

[0080] The experiment was performed according to the grouping and experimental methods in the experiment of detecting EMT-related markers in cells by immunofluorescence staining, and the fluorescence intensity of LC3Ⅱ / Ⅰ protein and p62 was detected. The results are shown in Figure 8 .

[0081] The results showed that after incubation with TGF-β2 and metformin, the fluorescence intensity of LC3II / LC3I increased significantly and the expression of p62 was downregulated, which was consistent with the WB results.

[0082] 10. Cell cycle detection

[0083] Cells were plated at 1×10 6 Cells were seeded in 6-well plates and divided into the following groups: control group: cells were cultured normally for 48 h; metformin-treated group: cells were treated with 5 mM metformin for 24 h, and then cultured normally for 24 h; cells were harvested and permeabilized with pre-cooled 75% ethanol at 4 °C overnight. Cells were then treated with 1 mg / ml RNase A at 37 °C for 30 min and stained with 50 μg / ml propidium iodide for 15 min in the dark. Cells were then analyzed by flow cytometry, and the results are shown in Fig. 9 .

[0084] The results showed that flow cytometry analysis of propidium iodide (PI)-stained cells showed that the number of cells in the G0 / G1 phase increased and the number of cells in the S phase decreased.

[0085] 11. Cell Apoptosis Detection

[0086] The grouping and treatment methods were similar to the above-mentioned cell cycle detection test, and the apoptosis of each group of cells was measured using the Annexin V-FITC / PE apoptosis kit (BD Biosciences, San Jose, CA, USA). The cells were collected (consistent with the cell cycle detection step) and washed with PBS buffer, and then resuspended in 100 μl binding buffer. Annexin V-FITC (5 μl) was then added, and the cell suspension was incubated in the dark for 5 minutes, and then incubated in the dark for another 15 minutes in the presence of 5 μl propidium iodide. The fluorescence intensity was measured by flow cytometry (FACSCalibur, BD Biosciences). The results are shown in Fig.10 .

[0087] The results showed that compared with the NC group, the cell apoptosis rate in the metformin-treated group was significantly increased, indicating that metformin could induce apoptosis of lens epithelial cells.

[0088] 12. Preparation of Metformin Eye Drops

[0089] C57BL / 6 mice were purchased from Liaoning Changsheng Biotechnology Co., Ltd. and maintained in an environment of 23°C, 45-55% humidity, and alternating day and night every 12 hours. Animals were killed by overdose of isoflurane. This study was approved by the Ethics Committee of the Aier Eye Institute of Central South University (IRB007), and the use and feeding of experimental animals followed the ARVO statement.

[0090] Nine-week-old male C57BL / 6 mice were anesthetized with 5% isoflurane, and the central anterior lens capsule was punctured at a depth of 2.5 mm from the corneal surface with a 31-gauge needle in the puncture group. Subsequently, 0.3% ofloxacin cream (Changshu Suzhou Fourth Pharmaceutical Co., Ltd.) and moxifloxacin hydrochloride (M828457, Shanghai McLean Biochemical Technology Co., Ltd., Shanghai, China) were injected. Mice in the metformin eye drops group were injected with 0.3% metformin (N886063, Shanghai McLean Biochemical Technology Co., Ltd., Shanghai, China) eye drops every 12 hours (8:00 and 20:00), and mice in the PBS group were given an equal amount of PBS as a control. The mice were euthanized on the second and fifth days, respectively, and the eyeballs were removed and fixed with 0.75% paraformaldehyde (PFA). 10 μm thick cryosections were obtained from a cryostat CM1860 (Leica, Heidelberg, Germany), transferred to slides, and incubated with blocking solution for 1 hour at room temperature. Subsequently, they were incubated with α-SMA antibody (1 / 100) (AF1032, Affinity Biosciences, Changzhou, China) in blocking solution at 4°C for 12 hours. The slides were washed with PBS and incubated with Cy3-labeled rabbit IgG fluorescent secondary antibody (1 / 200) and DAPI at room temperature for 1 hour. Images were acquired using microphotography systems BX53 and DP73 (OLYMPUS, Tokyo, Japan). The results are shown in Fig.11 .

[0091] The results showed that metformin eye drops had an effect on the expression of α-SMA after 2 days. Five days after acupuncture injury, the expression of α-SMA was weakened in the group given metformin eye drops compared with the PBS puncture group. In addition, after 5 days of uninterrupted administration of metformin eye drops, the fluorescence intensity of α-SMA was significantly reduced.

[0092] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. The use of metformin in the preparation of drugs for treating complications after cataract surgery.

2. The use according to claim 1, characterized in that: The postoperative complications of cataract surgery include posterior capsule opacity, abnormal proliferation of residual lens epithelial cells after surgery, hyperplasia of lens epithelial cells caused by migration, posterior capsule opacity and / or decreased vision after cataract surgery.

3. The use according to claim 1 or 2, characterized in that: The treatment includes: inhibiting epithelial-mesenchymal transition of human lens epithelial cells, inducing autophagy and / or apoptosis of lens epithelial cells, inducing G0 / G1 phase arrest of lens epithelial cells, regulating the expression of EMT-related markers, and regulating the expression of EMT-related pathway proteins.

4. The use according to claim 3, characterized in that: The inhibiting of epithelial-mesenchymal transition of human lens epithelial cells comprises: inhibiting cell viability, migration and / or proliferation of lens epithelial cells; The inducing lens epithelial cell autophagy comprises: increasing the expression level of LC3Ⅱ / Ⅰ protein and / or decreasing the expression level of p62 protein; The regulating the expression of EMT-related markers includes: reducing the expression levels of α-SMA and Fibronectin and / or increasing the expression level of the epithelial marker E-Cadherin; The EMT-related pathways include at least one of (1) to (3): (1) reducing the expression ratio of at least one of the following proteins: p-ERK / ERK, pB-RAF / B-RAF, p-SMAD / SMAD; (2) increase the ratio of p-AMPK / AMPK protein expression; (3) Increase the expression levels of p-AMPK and AMPK proteins.

5. The use according to any one of claims 1 to 4, characterized in that: The effective concentration of metformin is 2.5-40 mM.

6. The use according to claim 5, characterized in that: The effective concentration of metformin is 5-20 mM.

7. The use according to any one of claims 1 to 6, characterized in that: The administration method of metformin includes: ocular surface administration, intraocular administration or administration by preparing drug-loaded artificial lens.

8. A drug for preventing and treating complications of cataract, characterized in that: The invention comprises metformin and pharmaceutically acceptable excipients.

9. The drug according to claim 8, characterized in that The complications of cataracts include capsular opacification.

10. The drug according to claim 8 or 9, characterized in that Its dosage forms include drops, liquids, tablets or drug-loaded sustained-release intraocular lenses.

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