Use of theaflavin-3'-monogallate in inhibiting retinal pigment epithelial cell apoptosis and treating age-related macular degeneration

Abstract

The present invention discloses the use of theaflavin 3'-monogallate in inhibiting retinal pigment epithelial cell apoptosis and treating age-related macular degeneration (AMD), belonging to the field of biomedicine. Specifically, the invention discovers that theaflavin 3'-monogallate has the function of inhibiting retinal pigment epithelial cell apoptosis and promoting the inhibition of retinal pigment epithelial cell proliferation. Furthermore, the invention also discovers that theaflavin 3'-monogallate has the novel function of inhibiting AMD-like lesions caused by retinal pigment epithelial cell apoptosis. Therefore, it can be used to treat AMD, bringing good news to elderly blind patients in China.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the use of theaflavin-3'-monogallate in inhibiting retinal pigment epithelial cell apoptosis and treating age-related macular degeneration. Background Art

[0002] Age-related macular degeneration (AMD), also known as age-related macular degeneration (AMD), is an eye disease that causes irreversible vision loss or impairment due to degenerative changes in the retinal pigment epithelium (RPE) and retina. It is a common cause of blindness in people over 50 years old. Based on clinical manifestations and pathological features, AMD can be divided into atrophic AMD (dry form) and exudative AMD. Atrophic AMD manifests as subretinal drusen and geographic atrophy in the macula, while exudative AMD presents as choroidal neovascularization (CNV), retinal pigment epithelial detachment, and macular hemorrhage and edema.

[0003] The pathogenesis of AMD is complex and may be related to aging, metabolic changes, oxidative damage, light damage, immune inflammation, and genetic changes in retinal pigment epithelial cells. Epidemiological data show that dyslipidemia is not only a risk factor for atherosclerotic disease in middle-aged and elderly people, but also an important risk factor for AMD. Studies on the relationship between dyslipidemia and AMD have shown that low-density lipoprotein (LDL) can pass through Bruch's membrane through the fenestra of choroidal endothelial cells and reach the retinal pigment epithelium and outer retina. Over time, more and more lipids are deposited in Bruch's membrane, inducing apoptosis of retinal pigment epithelial cells. In addition, functional changes and apoptosis of retinal pigment epithelial cells are considered to be important early pathological changes in AMD. Therefore, dyslipidemia may be closely related to the occurrence and development of AMD. Other studies have shown that oxidized low-density lipoprotein (ox-LDL) activates the lectin-like oxidized low-density lipoprotein receptor type I (LOX-1), upregulating the expression of multiple inflammatory factors (TNFα, IL-1, IL-6, IL-8), adhesion molecules, and chemokines, thereby promoting the adhesion and aggregation of inflammatory cells and platelets in the choroidal vessels, thereby mediating the inflammatory response. Furthermore, ox-LDL can stimulate macrophages and retinal pigment epithelial cells to produce vasoactive factors such as vascular endothelial growth factor (VEGF), thereby promoting endothelial cell proliferation and migration and choroidal angiogenesis by promoting the production of early inflammatory factors such as TNFα and IL-1.

[0004] China is the birthplace of tea. As one of the seven necessities of life (firewood, rice, oil, salt, soy sauce, vinegar, and tea), tea drinking is not only a significant part of ancient and modern China's intangible cultural heritage. Medical research suggests that tea drinking can provide antioxidant benefits, regulate immunity, and inhibit aging. Further research has shown that catechins can inhibit telomerase shortening, thereby suppressing cellular aging. Tea can also reduce cognitive deficits and brain morphological changes associated with aging in mice.

[0005] Theaflavin compounds are a general term for a class of compounds with benzophenone structure formed during the processing of black tea, mainly including theaflavin (TF1, Figure 1 A), Theaflavin-3-gallate (TF2A, Figure 1 B), Theaflavin-3'-monogallate (TF2B, Figure 1C) and Theaflavin-3,3'-digallate (TF3, Figure 1 D) 4 types.

[0006] Studies have shown that theaflavins have pharmacological properties such as lowering blood pressure, regulating blood sugar, and acting as antioxidants and anti-tumors. Regarding cytoprotection, TF3 inhibits erastin-induced ferroptosis in osteoarthritis chondrocytes via the Nrf2 / GPX4 signaling pathway. Theaflavins, by regulating the AKT / mTOR / RPS6 signaling pathway, inhibit the overactivation of primordial follicles and follicular atresia, alleviating cyclophosphamide-induced ovarian damage. Regarding anti-aging, TF3 improves oocyte quality and regulates granulosa cell function by regulating the mTOR signaling pathway and autophagy, thereby delaying ovarian aging. By regulating the MAPK signaling pathway, theaflavins have therapeutic effects on various diseases. Studies have shown that theaflavins have a protective effect on the kidneys of type 2 diabetic mice, and this protective effect is achieved through regulating the MAPK signaling pathway. TF3 can regulate ROS-mediated ferroptosis and apoptosis in human osteosarcoma cells via the MAPK signaling pathway. TF2B exerts anti-inflammatory effects during influenza virus infection by regulating the TLR4 / MAPK / p38 pathway. TF2B increased the number of lymphocytes in the blood of mice infected with influenza virus and reduced the number of neutrophils, monocytes and platelets. TF2B also reduced the mRNA expression levels of inflammatory cytokines (IL-6, TNF-α and IL-1β), chemokines (CXCL-2 and CCL-3) and interferons (IFN-α and IFN-γ) after influenza virus infection. It downregulated the expression levels of TLR4, p-p38, p-ERK and cytokines IL-6, TNF-α, IL-1β and IL-10. In summary, theaflavins can protect cells and inhibit cell aging by regulating signaling pathways. However, there is still a lack of research at home and abroad on the protective effects of theaflavins on retinal pigment epithelial cells and the prevention and treatment of age-related macular degeneration. Summary of the Invention

[0007] In view of the above-mentioned deficiencies in the prior art, the present invention provides a new use of theaflavin-3'-monogallate, which can inhibit retinal pigment epithelial cell apoptosis, promote retinal pigment epithelial cell proliferation, and prevent and treat age-related macular degeneration.

[0008] To achieve the above-mentioned purpose, the technical solution adopted by the present invention to solve the technical problem is:

[0009] The use of theaflavin-3'-monogallate in promoting the proliferation of retinal pigment epithelial cells and inhibiting their apoptosis.

[0010] Use of the theaflavin-3'-monogallate in preparing a drug for treating diseases induced by retinal pigment epithelial cell apoptosis.

[0011] A preparation for preventing or treating age-related macular degeneration, comprising the above-mentioned theaflavin-3'-monogallate or its salt as an active ingredient, and pharmaceutically acceptable carriers and / or excipients.

[0012] Furthermore, the preparation is an oral preparation, an injection preparation, a spray or an ophthalmic preparation.

[0013] Furthermore, ophthalmic dosage forms include eye drops, eye ointments, eye sprays, eye gels, eye patches, intraocular injections, ophthalmic microspheres, ophthalmic implants, periocular injections or ophthalmic sustained-release preparations.

[0014] A pharmaceutical composition for treating age-related macular degeneration, comprising the above-mentioned theaflavin-3'-monogallate.

[0015] Beneficial effects of the present invention:

[0016] The present invention discovered that theaflavin-3'-monogallate has the function of inhibiting retinal pigment epithelial cell apoptosis and promoting the inhibition of retinal pigment epithelial cell proliferation. At the same time, it was also found that it has a new function of inhibiting AMD-like lesions caused by retinal pigment epithelial cell apoptosis. It can be used to treat age-related macular degeneration, bringing good news to Chinese elderly blind patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 These are the structural formulas of four theaflavins;

[0018] Figure 2 The effect of TF2B on the proliferation of ARPE-19 cells;

[0019] Figure 3 This is the RNA-seq result analysis diagram;

[0020] Figure 4 The role of MKP-1 in TF2B-regulated retinal pigment epithelial cell proliferation;

[0021] Figure 5 The role of Ox-LDL in TF2B-regulated MKP-1-mediated proliferation of retinal pigment epithelial cells;

[0022] Figure 6 TF2B reduces retinal pigment epithelial cell apoptosis by inhibiting MKP-1;

[0023] Figure 7 To test the protection of TF2B on mouse retinal epithelial cells;

[0024] Figure 8 The expression changes of pro-inflammatory cytokines and anti-inflammatory cytokines in mice. DETAILED DESCRIPTION

[0025] The specific embodiments of the present invention are described below to facilitate understanding of the present invention by those skilled in the art. However, it should be clear that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the appended claims, these changes are obvious, and all inventions and creations utilizing the concepts of the present invention are protected.

[0026] Example 1 Effects of theaflavin-3'-monogallate (TF2B) on apoptosis and proliferation of human retinal pigment epithelial cells (ARPE-19)

[0027] Human retinal pigment epithelial cells (ARPE-19) were purchased from ATCC and cultured in F12 medium containing 10% FBS. Cells entering the logarithmic growth phase were collected for experiments. Four theaflavins monomers were treated at four concentrations of 0.5μg / mL, 1.0μg / mL, 1.5μg / mL, and 2.0μg / mL for concentration-dependent cell proliferation experiments and morphological observations. Cells were treated with TF2B at a concentration of 1.5μg / mL with statistically significant differences, and cells were collected at 0h, 12h, 24h, 48h, and 72h for cell proliferation rate experiments and morphological observations.

[0028] (1) Cell morphology observation

[0029] ARPE-19 cells in each group at each time point were collected, cell smears were prepared, and the morphology of ARPE-19 cells in each group was observed under an ordinary optical microscope.

[0030] (2) MTT assay to detect retinal pigment epithelial cell viability

[0031] ARPE-19 cells were seeded in 96-well plates and treated at the corresponding time points for different groups. The culture medium was removed, and 0.5 mg / mL MTT was added and incubated at 37°C for 4 hours. After discarding the MTT, 150 μL of DMSO was added. The culture plates were shaken vigorously for 10 minutes in the dark, and the absorbance of the solution at 570 nm was measured using a microplate reader. Each experiment was repeated at least three times, and the data were collected. The average value was taken as the final value. The absorbance value of the control group was set as 100%, and the absorbance values ​​of the remaining groups were expressed as the percentage of the absorbance value of the control group.

[0032] To determine whether theaflavins have a proliferative effect on human retinal pigment epithelial cells (ARPE-19), we first conducted a dose-effect experiment on four theaflavins monomers (TF1, TF2A, TF2B, and TF3). Figure 2 .

[0033] like Figure 2 As shown in A, H2O2 significantly inhibited cell proliferation and promoted cell senescence. Meanwhile, TF1, TF2A, and TF3 did not significantly increase the proliferation of ARPE-19 cells, indicating that theaflavin (TF1), theaflavin-3-monogallate (TF2A), and theaflavin-3,3'-digallate (TF3) in black tea may not have a relevant effect on the inhibition of human retinal pigment epithelial cell proliferation caused by aging.

[0034] Theaflavin-3'-gallate (TF2B) at a concentration of 1.5 μg / mL could significantly promote cell proliferation (p<0.05, Figure 2 A). We further conducted a time-dependent experiment on the cell proliferation promoted by 1.5 μg / mL TF2B, setting five time points of 0, 12, 24, 48 and 72 hours to observe whether the cell proliferation was time-dependent. We observed that at 24 hours, the cells showed significant proliferation (p < 0.05, Figure 2 B). Therefore, our subsequent preliminary experiments used a concentration of 1.5 μg / mL for 24 hours to observe other indicators such as cell proliferation and apoptosis.

[0035] Example 2 Bioinformatics Analysis of Genes Regulated by TF2B in ARPE-19 Cells

[0036] Cells from the treatment group and the control group were collected and total RNA was isolated using the RNeasy kit (Qiagen, USA). RNA purity was checked using a nanophotometer spectrophotometer (Invitrogen, USA), and integrity was assessed using the RNA Nano 6000 detection kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, USA). Sequencing libraries were prepared using the NEBNext UltraTM RNA library preparation kit recommended by Illumina (NEB, USA). After cluster generation, the library was sequenced on the Illumina Novaseq platform and 150bp paired-end reads were generated. The raw reads were filtered for subsequent analysis by removing reads containing adapters, poly-N, and low-quality reads. Hisat2 v2.0.5 was used to build an index for the reference genome, and Hisat2 v2.0.5 was used to align the paired-end cleaned data with the reference genome. FeatureCounts v1.5.0-p3 was used to calculate the number of reads for each gene. The FPKM of each gene and the read count mapped to the gene were then calculated based on the length of the gene. Differentially expressed mRNAs were analyzed using the DESeq2 R package (1.16.1). Genes with adjusted P-values ​​< 0.05 identified by DESeq2 were classified as differentially expressed based on a negative binomial distribution test. Gene Ontology (GO) enrichment analysis of differentially expressed genes was performed using the ClusterProfiler R package, which corrects for gene length bias. GO terms with adjusted P-values ​​< 0.05 were considered significantly enriched by differentially expressed genes. The statistical enrichment of differentially expressed genes within the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways was tested using the R package Statistical Analysis.

[0037] After confirming that TF2B, a compound found in theaflavins, regulates human retinal pigment epithelial (ARPE-19) cells, we collected and extracted total RNA from control and treatment groups and performed sequencing analysis. Through sequencing, enrichment analysis, and visualization, we identified MKP-1 as a gene that may be regulated by TF2B in ARPE-19 cells. Furthermore, MKP-1 was downregulated in cells treated with TF2B.

[0038] Example 3 TF2B promotes proliferation by regulating MKP-1

[0039] 1. Lentivirus overexpression of MKP1 protein (DUSP1 gene)

[0040] pLenti-GIII-CMV-DUSP1 (#18670061) was purchased from Applied Biological Materials Inc. (Canada). The plasmid was transformed in Escherichia coli DH5α cells by heat shock, spread on LB agar plates, selected with antibiotics and amplified by LB medium. To produce lentiviral particles, 80% confluent 293T cells were infected with the above 10 μg of lentivirus and plasmids containing VSV-G envelope glycoprotein (3.5 μg), Rev (2.5 μg) and ΔR8.74 packaging protein (6.5 μg) by polybrene (Sigma, USA). 48 hours after infection, the supernatant rich in lentiviral particles was harvested, filtered, and stored frozen at -80°C for further use. The cells were cultured for 24 hours before infection, and then the lentivirus carrying DUSP-1 was infected with the cells by polybrene.

[0041] MKP-1 knockdown

[0042] MKP-1 / DUSP1 siRNA was purchased from Santa Cruz (catalog number sc-35938, Santa Cruz, California, USA). 4 Cells were seeded in 6-well plates and cultured overnight, then switched to serum-free medium for 4 hours. 4 μl of DharmaFECT2 (Thermo Scientific, USA) was added to the serum-free medium and incubated at room temperature for 5 minutes. The mixture was then mixed with 5 pM MKP-1 siRNA and incubated for 20 minutes. Finally, the mixture was added to the cells along with serum-containing medium. Cells were harvested 72 hours later for immunoblotting and fluorescent staining.

[0043] 3. Western Blot

[0044] Cells from each group were harvested using a cell scraper and placed in RIPA buffer. Protein was quantified using a BCA protein quantification kit (Biyuntian, Suzhou, Jiangsu). Based on the protein content of each sample, a volume of 10 μg of protein was used as the loading volume. Equal amounts of protein from the treatment and control groups were subjected to SDS-PAGE gel electrophoresis. Before loading, the protein was denatured. A mixture of 1 volume of protein and 1 / 4 volume of loading buffer was prepared. The mixture was boiled at 95°C for 5 minutes, cooled in ice water, and centrifuged at 1000 rpm for 30 seconds. A 10 ng sample was heated at 95°C for 10 minutes before loading. Electrophoresis was performed for 2 hours using SADS-PAGE running buffer. The protein was then transferred to a PVDF membrane. Blocking buffer (5% BSA) was added, and the membrane was gently shaken on a shaker for 1 hour at room temperature. A polyclonal rabbit anti-human MKP-1 antibody (Abcam, USA, Cat. No. ab61201) was added and incubated overnight at 4°C. The next day, the membrane was washed three times with TBS buffer containing 0.1% Tween-20 for 10 minutes each time. Then, the membrane was incubated with HRP-coupled goat anti-rabbit IgG antibody at room temperature for 2 hours. The membrane was then washed three times with TBS buffer containing 0.1% Tween-20 for 10 minutes each time. ECL luminescence working solution was prepared by mixing according to the proportions in the instructions, and the PVDF membrane was placed in the ECL luminescence working solution until it was completely immersed. The membrane was incubated with shaking at room temperature for 5 minutes. After laminating with film, it was exposed in a dark room. After being treated with developer and fixer, it was rinsed with water and dried, and then the gel image was scanned and analyzed. After the antibodies bound to the membrane were eluted with membrane elution solution, blocking solution (5% BSA) was added and blocked at room temperature for 1 hour. Monoclonal mouse anti-human β-actin antibody (Hua'an Bio, China) was added and incubated overnight at 4°C. The next day, the membrane was washed three times with TBS buffer containing 0.1% Tween-20 for 10 minutes each time. Then, incubate with HRP-conjugated goat anti-mouse IgG antibody at room temperature for 2 hours. Wash three times with TBS buffer containing 0.1% Tween-20 for 10 minutes each. Prepare ECL working solution according to the instructions. Place the PVDF membrane in the ECL working solution until completely immersed. Incubate with horizontal shaking at room temperature for 5 minutes. Lay the membrane on film and expose it in a darkroom. After processing with developer and fixer, rinse with water and air dry, then scan and analyze the gel image.

[0045] 4. Immunofluorescence staining

[0046] BrdU was added to the culture medium of each cell group to a final concentration of 0.8 mg / mL (BrdU powder purchased from Sigma-Aldrich, purity ≥99% by HPLC). After 72 hours, the culture medium was discarded and the cells were fixed with 4% PFA for 30 minutes at room temperature. The 4% PFA was discarded, and the cells were permeabilized with 1% Triton-X 100 for 10 minutes at room temperature. The Triton-X 100 was discarded, and the cells were washed two to three times with PBS for 5 minutes each. The PBS was discarded, and the cells were blocked with 5% BSA for 1 hour. The primary antibody (rabbit polyclonal Cystatin C antibody, 1:100 dilution; Cat. No. ab226841, Abcam, Cambridge, MA, USA) was added and incubated overnight at 4°C. Wash sections three times with PBS for 3 minutes each. After blotting with absorbent paper, the corresponding secondary antibody, Cy5-conjugated goat anti-rabbit IgG (1:500 dilution; Cat. No. 111-175-144, Jackson Laboratories, USA), was added dropwise and incubated at room temperature in the dark for 2 hours. Wash sections three times with PBS for 5 minutes each. Discard the PBS, and add the primary antibody (rabbit monoclonal BrdU antibody, 1:100 dilution; Cat. No. ab6326, Abcam, Cambridge, MA, USA) to each well. Incubate cells overnight at 4°C. Wash sections three times with PBS for 3 minutes each. After blotting with absorbent paper, the corresponding secondary antibody, FITC-conjugated goat anti-rabbit IgG (1:500 dilution; Cat. No. 111-095-003, Jackson Laboratories, USA), was added dropwise and incubated at room temperature in the dark for 2 hours. Wash sections three times with PBS for 5 minutes each. Discard PBS, add 1 μg / mL DAPI to each well for staining, discard DAPI, wash three times with 1 mL PBS for 5 minutes each, and discard PBS. Mount the slides with mounting solution containing an anti-fluorescence quencher, and observe and collect images under a fluorescence microscope.

[0047] First, cells in the treatment group and the control group were labeled with BrdU. Then all cells were given H2O2 to induce senescence. Immunofluorescence cell staining was used to immunolabel BrdU, which marks proliferation, and Cystatin C, which marks retinal pigment epithelial cells for AMD. DAPI was also used to label the cell nuclei. The results are shown in Figure 2. Figure 4 .

[0048] like Figure 4 As shown in A, all cells showed AMD-like lesions, and the proliferation of cells in the control group was significantly inhibited, while cells treated with TF2B showed significant proliferation, indicating that it can neutralize the growth inhibition caused by senescence. Next, we purchased the MKP-1 siRNA library and effectively inhibited the expression of MKP-1 protein ( Figure 4B). Immunofluorescence staining results showed that cell proliferation increased after MKP-1 inhibition, indicating that MKP-1 activation led to cell proliferation inhibition and subsequent AMD-like lesions, and TF2B could significantly inhibit MKP-1 expression, thereby promoting the proliferation of retinal pigment epithelial cells ( Figure 4 C). At the same time, we conducted a rescue experiment. We constructed a lentiviral vector that overexpressed MKP-1 and transfected cells ( Figure 4 D). As shown in the figure, overexpression of MKP-1 in the control group completely halted cell proliferation. Even with TF2B administration, retinal pigment epithelial cell proliferation was significantly reduced compared to the non-overexpression group, indicating that TF2B-induced cell proliferation is achieved through inhibition of MKP-1.

[0049] 5. TF2B may inhibit MKP-1 by regulating ox-LDL, thereby promoting retinal pigment epithelial cell proliferation

[0050] Many studies have shown that theaflavins can lower blood lipids, and that oxidized low-density lipoprotein (ox-LDL) plays an important role in the progression of AMD. Therefore, we conducted experiments to explore the relationship between TF2B, ox-LDL, and MKP-1 and retinal pigment epithelial cell proliferation.

[0051] like Figure 5 As shown in A, TF2B significantly promoted ARPE-19 cell proliferation, but after adding commercially purchased ox-LDL, cell proliferation was significantly inhibited. Since TF2B promotes cell proliferation by inhibiting MKP-1, Figure 5 As shown in B, there was no significant change in cell proliferation between the knockdown MKP-1 group and the control group. However, when MKP-1 was overexpressed, the excess MKP-1 in the cells could neutralize the inhibitory effect of TF2B on MKP-1, resulting in a decrease in cell proliferation even after the addition of TF2B ( Figure 5 C) The above figure demonstrates that TF2B's pro-proliferative effect on AREP19 cells is mediated by MKP-1. Furthermore, the project team observed changes in cell proliferation following the simultaneous addition of ox-LDL and TF2B. Because TF2B regulates cell proliferation by inhibiting ox-LDL, the addition of ox-LDL prevents TF2B from reducing intracellular ox-LDL levels.

[0052] Therefore, compared to cells treated with TF2B alone and without ox-LDL, cells treated with both ox-LDL and TF2B showed increased intracellular ox-LDL levels and neutralized the proliferation-promoting function of TF2B. As shown in Figure B, cell proliferation was inhibited. However, when MKP-1 gene silencing was achieved by adding siRNA to the cells, the increased intracellular ox-LDL failed to inhibit cell proliferation by promoting the MAPK signaling pathway, where MKP-1 is involved. Consequently, cell proliferation was enhanced in cells treated with TF2B, ox-LDL, and MKP-1 siRNA simultaneously.

[0053] In cells with MKP-1 knockdown, there was no significant difference in cell proliferation regardless of whether ox-LDL was added, indicating that MKP-1 is a downstream gene regulated. Figure 6 As shown in Figure C, overexpression of MKP-1 significantly reduced cell proliferation even with the addition of TF2B, indicating that TF2B promotes cell proliferation by regulating MKP-1. However, the addition of ox-LDL significantly inhibited cell proliferation. When TF2B and ox-LDL were added simultaneously, cell proliferation was also inhibited in cells overexpressing MKP-1. This rescue experiment demonstrates that TF2B indirectly inhibits MKP-1 by inhibiting ox-LDL, thereby promoting cell proliferation.

[0054] 6. TF2B inhibits retinal pigment epithelial cell apoptosis under inflammatory conditions

[0055] The apoptosis of human retinal pigment epithelial cells (ARPE-19) was detected using the Annexin V-PI staining kit from Yisheng. 5×10 5 cells. Resuspend the cells in 500 μL of 1X Annexin V Binding Buffer to the supernatant. Then, add 5 μL of Annexin V-FITC and 5 μL of propidium iodide (PI). Incubate at room temperature for 5 minutes in the dark. Analyze annexin V-FITC binding using a FITC signal detector on a flow cytometer (excitation 488 nm; emission 350 nm). Simultaneously, analyze PI staining using a phycoerythrin emission signal detector.

[0056] Existing literature has shown that the occurrence and development of AMD is closely related to inflammatory cytokines and the associated aging of retinal pigment epithelial cells. When exploring the mechanism of TF2B in treating AMD, we paid close attention to the relationship between TF2B and cell apoptosis. Figure 6As shown in A, under the action of the proinflammatory cytokine TNF-α, ARPE-19 cell apoptosis significantly increased, but after administration of TF2B, apoptotic ARPE-19 cells were significantly reduced. Knockdown of MKP-1 will promote the reduction of apoptosis to a certain extent, but after administration, the expression of MKP-1 can be completely inhibited, thereby reducing cell apoptosis ( Figure 6 B). We then performed a rescue experiment by overexpressing MKP-1 and observed a significant increase in cell apoptosis. Even with the administration of TF2B, it was not able to counteract the excessive MKP-1 in the cells, and thus a large number of cells still underwent apoptosis ( Figure 6 C) The above preliminary experiments indicate that under inflammatory conditions, TF2B regulates retinal pigment epithelial cell apoptosis by inhibiting MKP-1.

[0057] Example 4 TF2B inhibits AMD-like lesions in mouse retinal pigment epithelial cells

[0058] 1. In vitro and in vivo experiments

[0059] (1) The ATG start codon of Dusp-1 (Mkp-1) is located in exon 1. Using CRISPR / Cas9 technology, sgRNA (TTGGGCTGTGTGTGCGACG) was designed for Dusp-1 exon 1. Cas9, sgRNA, and targeting vector were co-injected into fertilized eggs by high-throughput electroporation to obtain Dusp-1 gene conditional knockout mice. Genomic DNA was extracted from the mouse tail tissue, and then PCR amplification was performed to identify positive mice. Positive Dusp-1 f / + Mice were mated with wild-type C57BL / 6J mice to obtain F1 heterozygotes, which were then self-fertilized to obtain F2 homozygous Dusp-1 f / f Furthermore, the F2 homozygotes were mated with Vmd-Cre mice and then self-fertilized to obtain Vmd-Cre; Dusp-1 f / f (Referred to as MKP-1-KO mice). The control group of mice was Vmd-Cre; Dusp-1 f / + mouse.

[0060] Table 1. PCR primer sequences

[0061]

[0062] (2) Injection of lentivirus into the tail vein of mice to overexpress MKP1 protein

[0063] The lentiviral particles enriched by expression in 293T cells in the previous step were filtered through a 0.45 μm filter, subjected to ultracentrifugation (113,000 × g for 2 h at 4°C) to a 100-fold concentration, and stored at −80°C. The lentiviral supernatant was then thawed at 37°C and diluted with 0.9% saline (Sichuan Kelun Pharmaceutical Co., Ltd., Chengdu, Sichuan, China) and polybrene (8 μg / mL final concentration; Sigma-Aldrich, USA) in a 50 μl injection volume (containing 1.6 × 10 7 The virus was intravenously injected into the tail vein of C57BL / 6J mice (n=6) once every 3 days for 3 consecutive injections.

[0064] (3) Immunofluorescence staining

[0065] Paraffin sections were cleared and hydrated [xylene I (10 min), xylene II (10 min), xylene III (10 min), ethanol I (10 min), ethanol II (10 min), ethanol III (10 min), 95% ethanol (10 min), 75% ethanol (10 min), 50% ethanol (10 min), PBS (10 min)] and subjected to citric acid antigen retrieval for 10 min. The sections were blocked with goat serum for 2 hours. The serum was discarded, and the primary antibody (rabbit monoclonal Cathepsin B antibody, 1:100 dilution; cat. no. ab214428, Abcam, Cambridge, MA, USA) was added and incubated overnight at 4°C. Wash sections three times with PBS for 5 minutes each. After blotting with absorbent paper, add the corresponding secondary antibody, Cy5-conjugated goat anti-rabbit IgG (1:500 dilution; Cat. No. 111-175-144, Jackson Laboratories, USA), and incubate at room temperature in the dark for 2 hours. Wash sections three times with PBS for 5 minutes each. Mount sections with anti-quencher mounting solution and observe and acquire images under a fluorescence microscope.

[0066] (4) Since the results of in vitro experiments cannot fully represent the actual situation in vivo, we conducted a preliminary experiment in mice. First, we constructed Mkp-1 conditional knockout (Mkp-1 CKO) mice using CRISPR / Cas9 technology, and then obtained retinal epithelial cell-specific Mkp-1 knockout mice by mating with Cre mice and then self-fertilizing. Figure 7 A).

[0067] Using 12-month-old (old) mice as pre-experimental subjects, we observed a significant decrease in the expression of Cathepsin B (CatB), a marker of retinal function, in the model group after inducing AMD in mice with hydroquinone and strong light, while the expression of Cat B in TF2B mice injected with 40 mg / kg / d via the tail vein was significantly increased ( Figure 7 B).

[0068] Under the same induction method, the expression of Cat B in Mkp-1 CKO mice was higher than that in isotype control mice, indicating that MKP-1 protein may be one of the factors that induce AMD. After TF2B administration, the expression of Cat B was significantly increased in both Mkp-1 CKO mice and isotype control mice ( Figure 7 C). In the rescue experiment, we injected mice with a lentivirus that overexpressed MKP-1. We observed that Cat B expression was significantly reduced in mice overexpressing MKP-1, and even the administration of TF2B could not restore the damage to retinal pigment epithelial cell function caused by MKP-1 overexpression ( Figure 7 D) Therefore, three animal models were used: aged mice, Mkp-1 CKO mice, and MKP-1 overexpressing mice, to preliminarily verify that TF2B inhibits AMD-like lesions in retinal pigment epithelial cells through both positive and negative approaches.

[0069] 2. TF2B regulates AMD-like lesions in mice by regulating phasic regulation

[0070] Mouse serum was collected, and the expression changes of pro-inflammatory cytokines and anti-inflammatory cytokines in the serum of mice in each group were detected using mouse serum IL1-β (Abcam, USA, Catalog No. ab197742), IL-6 (Abcam, USA, Catalog No. ab222503), TNF-α (Abcam, USA, Catalog No. ab100747), and IL-10 kits (Abcam, USA, Catalog No. ab108870).

[0071] Based on in vitro experiments, we speculated that ox-LDL may be related to TF2B-mediated protection of retinal pigment epithelial cells. In the in vivo experiments, we observed the changes in pro-inflammatory cytokines and anti-inflammatory cytokines in the serum of mice between the treatment group and the control group. Figure 8As shown, TF2B administration significantly reduced serum levels of proinflammatory cytokines (IL-1β, IL-6, and TNF-α) and significantly increased levels of anti-inflammatory cytokines (IL-10). When mice were fed a high-fat diet to elevate ox-LDL cholesterol, serum levels of proinflammatory cytokines increased significantly, whereas TF2B administration reduced these levels. MKP-1 knockout reduced proinflammatory cytokines and increased anti-inflammatory cytokines, and this effect was not affected by the ox-LDL increase induced by the high-fat diet, suggesting that ox-LDL-induced proinflammatory cytokine expression is mediated by MKP-1. TF2B's effect on reducing proinflammatory cytokines is achieved by inhibiting ox-LDL and, consequently, MKP-1. Therefore, when MKP-1 was knocked out, there was no significant difference in inflammatory cytokine expression between the TF2B-treated and control groups. When MKP-1 is overexpressed, serum levels of proinflammatory cytokines are significantly elevated, but TF2B administration can reduce these levels to a certain extent. Moreover, when a high-fat diet brings about a large amount of ox-LDL and MKP-1 is overexpressed, TF2B can inhibit the expression of MKP-1 to a certain extent by inhibiting ox-LDL, thereby reducing pro-inflammatory cytokines and increasing anti-inflammatory cytokines.

[0072] Finally, it should be noted that the above specific implementation methods are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. Use of theaflavin-3'-monogallate in the preparation of a medicament for preventing or treating age-related macular degeneration.

2. The use according to claim 1, characterized in that The drug can promote the proliferation of retinal pigment epithelial cells and inhibit their apoptosis.

3. The use according to claim 1, wherein the drug further comprises theaflavin-3'-monogallate in a pharmaceutically acceptable carrier and / or excipient.

4. The use according to claim 1, characterized in that The medicine is in the form of oral preparation, injection preparation, spray or ophthalmic preparation.

5. The use according to claim 4, characterized in that The ophthalmic dosage forms include eye drops, eye ointments, eye sprays, eye gels, eye patches, intraocular injections, ophthalmic microspheres, ophthalmic implants, periocular injections or ophthalmic sustained-release preparations.

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