Use of thrombospondin 1 in the preparation of a medicament for preventing and / or treating retinal damage

Abstract

The application provides application of thrombospondin 1 in preparation of a drug for preventing and / or treating retinal injury, and belongs to the technical field of eye disease drugs. The application verifies the anti-inflammatory effect of thrombospondin 1 (THBS-1) in experimental blue light induced retinal inflammation by means of in vitro cell experiments and in vivo animal experiments, and verifies the role of thrombospondin 1 in inhibiting pathological neovascularization and the protective effect on retinal injury. Therefore, the application provides application of thrombospondin 1 in preparation of a drug for preventing and / or treating age-related macular degeneration.

Description

[0001] This application is a divisional application of the Chinese patent application No. 202011158055.7, filed on December 11, 2020, entitled "Application of Thrombospondin 1 in Preparation of Drugs for Preventing and / or Treating Age-related Macular Degeneration", the whole content of which is incorporated herein by reference. TECHNICAL FIELD

[0002] The present application belongs to the technical field of eye disease drugs, and particularly relates to application of thrombospondin 1 in preparation of drugs for preventing and / or treating retinal injury. BACKGROUND

[0003] Retinal degenerative diseases mainly include age-related macular degeneration (AMD), diabetic retinopathy and retinitis pigmentosa, etc., and are one of the main causes of impaired vision worldwide. AMD is the most common form of retinal degenerative disease, which is mainly divided into two forms: non-neovascular AMD (dry AMD) and neovascular AMD (wet AMD). Dry AMD is related to the damage and death of retinal pigment epithelial cells (RPE) and the degeneration of photoreceptors, while wet AMD is usually attributed to the formation of choroidal neovascularization (CNV), which seriously affects the vision of patients.

[0004] Retinal inflammation is an important pathogenic process of AMD, and RPE dysfunction plays a crucial role in retinal inflammation and CNV formation during the progression of AMD. As a bridge between systemic blood circulation and neural retina, retinal pigment epithelial cells are responsible for material exchange between the retina and adjacent tissues, maintaining the balance between inflammatory and angiogenic responses in the subretinal space. When RPE is damaged by adverse stimuli such as blue light, the immune / vascular will be destroyed, leading to pathological inflammation and / or angiogenic response in the retina.

[0005] At present, the main treatment method for retinal inflammation in AMD and other retinal degenerative diseases is intravitreal injection of triamcinolone acetonide, which can effectively inhibit retinal edema and inflammatory cell infiltration caused by inflammation. However, complications such as hormone-induced glaucoma and acute endophthalmitis are very common. Anti-vascular endothelial growth factor (VEGF) therapy is recognized as the most primary treatment for neovascular AMD in clinical practice. However, a large number of clinical patients do not respond to anti-VEGF therapy, with a proportion as high as 45%. In addition, in clinical practice, anti-inflammatory (triamcinolone acetonide) and anti-vascular treatments are usually used in combination to achieve satisfactory treatment effect for AMD. Therefore, a promising adjuvant or alternative therapy in parallel with the current treatment strategy is urgently needed. SUMMARY

[0006] Therefore, the application aims to provide an application of thrombospondin 1 in preparation of a drug for preventing and / or treating retinal injury.

[0007] The application provides an application of thrombospondin 1 in preparation of a drug for preventing and / or treating retinal injury.

[0008] Preferably, the retinal injury comprises retinal pigment epithelial cell injury.

[0009] Preferably, the retinal injury is induced by blue light.

[0010] Preferably, the amino acid sequence of the thrombospondin 1 is shown in the sequence table SEQ ID NO: 1.

[0011] The application provides an application of thrombospondin 1 in preparation of a drug for preventing and / or treating retinal injury. The application proves that thrombospondin 1 has a regulating effect on the pathological process of retinal injury from the cell level and the animal level. THBS-1 protects the retina from blue light-induced retinal injury, and the protective effect of THBS-1 on blue light-induced retinal injury in vitro and in vivo shows that, in vivo, the proliferation ability and survival rate of blue light irradiated retinal pigment epithelial cells (RPE) are obviously recovered through THBS-1 treatment, and the decrease of the expression level of tight junction proteins (Occludin and ZO-1) in RPE cells representing the function of RPE cells under blue light irradiation is recovered through THBS-1 treatment. These results prove that THBS-1 has a protective effect on blue light-induced RPE injury. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 Blue light irradiation induces abnormal levels of inflammatory factors in ARPE-19 cells, wherein Figure 1 A shows that the mRNA expression levels of pro-inflammatory cytokines MCP-1, TNF-α, IL-1β, IL-6 and IL-8 are significantly up-regulated after blue light irradiation, and the expression level of the immunosuppressive cytokine TGF-β1 is down-regulated; Figure 1 B shows that the change of protein expression level is consistent with the change of mRNA expression, and β-actin is used as an internal reference protein; error bar: mean ± standard deviation; **P<0.01, Student's t test; BL: blue light;

[0013] Figure 2 Blue light induces the expression of inflammatory factors by activating the NF-κB pathway in ARPE-19 cells, wherein Figure 2 A shows that the phosphorylation of IRAK-1, c-Jun, STAT3 and p38 in the protein expression level of the core transcription factor involved in the retinal inflammatory pathway does not increase;Figure 2 B represents that the expression level of NF-κB p65 protein in the nucleus of the BL group is increased, but the expression level of cytoplasmic NF-κB p65 protein is lower compared with the control group by detecting the up-regulation of the phosphorylation level of IκBα by western blot; the expression level of nuclear protein is taken as the internal reference protein of histone H3, and the expression level of cytoplasmic protein is taken as the internal reference protein of β-actin; Figure 2 C represents that immunostaining shows that, after blue light irradiation, NF-κB p65 occurs nuclear translocation (as shown by red arrows); Figure 2 D represents that the western blot results show that BAY 11-7082 can significantly reduce the expression levels of MCP-1, TNF-α, IL-1β, IL-6 and IL-8, and BAY 11-7082 cannot reverse the down-regulation of TGF-β1. Error bar: mean ± standard deviation; **P<0.01, Student's t test; BL: blue light; Scale bar: 50 μm;

[0014] Figure 3 THBS-1 inhibits the retinal inflammation caused by blue light by inhibiting the NF-κB signaling pathway; wherein Figure 3 A represents the protein expression levels of NF-κB p65 and P-IκBα; THBS-1 treatment inhibits the phosphorylation of IκBα and the up-regulation of NF-κB p65 expression in nuclear protein in blue light-induced RPE cells. As Figure 3 B represents that the qPCR results and Figure 3 C represents that, as shown by the western blot analysis, the expression levels of MCP-1, TNF-α, IL-1β, IL-6 and IL-8 induced by blue light are significantly reduced by THBS-1 treatment, and THBS-1 treatment can rescue the reduced expression of TGF-β1; Figure 3 D is to perform immunostaining to detect the expression of MCP-1 (monocyte chemotactic protein), which is mainly distributed in the RPE layer; THBS-1 treatment significantly reduces the increase in the expression of MCP-1 induced by blue light; Figure 3 E is to perform immunostaining to detect the expression of CD68 (a marker of monocytes); CD68-positive cells are observed in the BL group, and THBS-1 treatment can reduce the chemotaxis of CD68-positive cells; Figure 3 F is a schematic diagram of THBS-1 inhibiting the retinal inflammation caused by blue light by inhibiting the NF-κB signaling pathway; error bar: mean ± standard deviation; **P<0.01, Student's T test; BL: blue light; INL: inner nuclear layer; ONL: outer nuclear layer; RPE: retinal pigment epithelial layer; scale bar: 50 μm;

[0015] Figure 4To activate the angiogenic response of hMEC-1 cells by BL-CM, human microvascular endothelial cells (hMEC-1) were treated with BL-CM; changes in the proliferation, migration and tube formation abilities of hMEC-1 cells were observed; wherein Figure 4 A is the EdU result, the ratio of EdU-positive cells increased after hMEC-1 cells were treated with BL-CM; Figure 4 B shows the results of scratch test and Figure 4 C shows the results of Transwell test, showing that the horizontal and vertical migration abilities of hMEC-1 cells in the BL-CM group were enhanced; Figure 4 D is the tube formation test result, the number of lumen formation and the number of branches and nodules in the BL-CM group were significantly higher than those in the control group; BL: blue light; BL-CM: conditioned medium of ARPE-19 cells irradiated by blue light; scale bar: 100 μm Figure 4 A, Figure 4 D); 200 μm Figure 4 B, Figure 4 C);

[0016] Figure 5 THBS-1 inhibited the VEGF-mediated angiogenic response in hMEC-1 cells, wherein Figure 5 A is the EdU test result of hMEC-1 cells treated with THBS-1 and VEGF or without treatment; Figure 5 B is the quantitative analysis result, showing that THBS-1 inhibited the promotion of VEGF on cell proliferation; Figure 5 C is the horizontal migration of hMEC-1 cells for 48 hours evaluated by scratch test; Figure 5 D is the quantitative result of scratch test migration rate; Figure 5 E is the vertical migration of hMEC-1 cells for 24 hours analyzed by Transwell test; Figure 5 F is the Transwell test migration rate (%) result evaluated by cell counting; the results of the two migration tests show that THBS-1 inhibited the VEGF-mediated migration; Figure 5 G is the tube formation test analysis result; Figure 5 H Figure 5 J shows that the number of tube formation nodules, branches and lumens in five random fields of each group were quantified, and the results show that THBS-1 inhibited the promotion of VEGF on the tube formation ability of hMEC-1 cells; error bar: mean ± standard deviation; *P<0.05, **P<0.01, one-way ANOVA followed by LSD post-hoc test; scale bar: 100 μm Figure 5 A, Figure 5 G); 200 μm Figure 5 C, Figure 6 E);

[0017] Figure 6 To inhibit the formation of new blood vessels in vivo for THBS-1, wherein Figure 6 A indicates the expression level of VEGF evaluated by immunostaining; in mice exposed to blue light, a clear expression of VEGF was observed around the retinal RPE layer, while intravitreal injection of THBS-1 reduced the expression level of VEGF compared to control mice; the laser-induced CNV model was used to evaluate the ability of THBS-1 to inhibit the formation of choroidal neovessels in vivo, Figure 6 B is the spectral domain optical coherence tomography (SD-OCT) performed 7 days after the establishment of the laser-induced CNV model and Figure 6 C is the fluorescein angiography (FA) and was detected again 7 and 14 days after administration; the red arrows point to the CNV area; THBS-1 was effective in reducing neovessels, comparable to anti-VEGF; Figure 7 D indicates that the quantitative analysis of fluorescein leakage showed that the reduction of the area of CNV vascular leakage with THBS-1 treatment was comparable to that with anti-VEGF treatment (n = 6); error bars: mean ± standard deviation; *P <0.05, **P <0.01, one-way ANOVA followed by LSD post-hoc test; BL: blue light; INL: inner nuclear layer; ONL: outer nuclear layer; RPE: retinal pigment epithelial layer; scale bar: 50 μm;

[0018] Figure 7 To the effect of blue light on the tight junction indicators of ARPE-19 cells, wherein Figure 7 A is the mRNA expression level of tight junction proteins (Occludin and ZO-1) in ARPE-19 cells was significantly down-regulated after blue light irradiation results; Figure 8 B indicates that immunocytochemical analysis showed down-regulation of protein expression of Occludin and ZO-1 in RPE cells after blue light irradiation compared to control cells; error bars: mean ± standard deviation; **P <0.01, Student's t-test; BL: blue light; scale bar: 50 μm;

[0019] Figure 8 To prevent retinal damage caused by blue light at in vivo and in vitro levels for THBS-1, wherein Figure 8 A indicates that the EdU results show that THBS-1 can significantly rescue the proliferative capacity of RPE cells inhibited by blue light; Figure 8 B indicates that the results of live / dead staining show that THBS-1 rescues the decrease in RPE cell viability induced by blue light; Calcein AM (live cells); PI (dead cells); Figure 8C represents the results of immunostaining with antibodies against Occludin and ZO-1, which showed that THBS-1 treatment could significantly restore the reduced expression of Occludin and ZO-1 induced by blue light; Figure 8 D represents the images of representative H&E-stained retinal sections obtained for each group, which showed that THBS-1 treatment could significantly alleviate the sparse nuclear distribution and depigmentation of the RPE layer after light exposure (as shown in the detailed images); Figure 8 E represents the measurement of the thickness of ONL at eight positions selected from the lower and upper halves of the optic nerve (n = 6); THBS-1 could significantly rescue the ONL thickness of the BL group; Figure 8 F represents the scotopic ERG, which showed that THBS-1 treatment (n = 6) rescued the reduced amplitude of a-wave and b-wave in the ERG after exposure to light; Figure 8 G represents the photopic ERG; Figure 8 H represents the scotopic amplitude results; Figure 8 I represents the photopic amplitude results; error bar: mean ± standard deviation; *P < 0.05, **P < 0.01, one-way ANOVA followed by LSD post-hoc test; BL: blue light; INL: inner nuclear layer; ONL: outer nuclear layer; RPE: retinal pigment epithelial layer; scale bar: 100 μm Figure 8 A, Figure 1 B); 50 μm Figure 1 C, Figure 2 D). DETAILED DESCRIPTION

[0020] The application provides application of thrombospondin 1 in preparation of a medicine for preventing and / or treating age-related macular degeneration.

[0021]

[0022] In the present application, the age-related macular degeneration (AMD) preferably includes non-neovascular age-related macular degeneration (dry AMD) and neovascular age-related macular degeneration (wet AMD). Dry AMD is associated with damage and death of retinal pigment epithelial cells (RPE) and degeneration of photoreceptors, and wet AMD is usually attributed to the formation of choroidal neovascularization (CNV), which seriously affects the vision of patients. The results show that thrombospondin 1 (THBS-1) shows more obvious efficacy than anti-VEGF treatment in inhibiting pathological angiogenesis, and THBS-1 has the potential to become an adjuvant therapy or alternative therapy to the current treatment regimen.

[0023] In the present application, an important pathogenic process of AMD also includes retinal inflammation, when retinal pigment epithelial cells are damaged, leading to pathological inflammation and / or angiogenic response of the retina. Experimental results show that THBS-1 has good protective effect on blue light-induced retinal damage in vitro and in vivo. At the same time, the anti-inflammatory function of THBS-1 is not possessed by anti-VEGF drugs, and its dual anti-inflammatory and anti-vascular function can avoid unnecessary burden on patients.

[0024] In the present application, the use dose of thrombospondin 1 per eye is preferably not less than 1.2 ng / g body weight of mice. In in vitro studies, the use concentration of thrombospondin 1 is not less than 4 nM.

[0025] The present application provides the use of thrombospondin 1 in the preparation of a medicament for preventing and / or treating retinal inflammation.

[0026] In the present application, the retinal inflammation is preferably induced by blue light. The method for preventing and / or treating the retinal inflammation preferably includes reducing the expression of MCP-1 and CD68 in the retina. In animals, MCP-1 (monocyte chemoattractant protein) and CD68 (inflammatory monocyte marker) are highly expressed in the mouse retina induced by blue light, and are significantly reduced after treatment with THBS-1.

[0027] The present application provides the use of thrombospondin 1 in the preparation of a medicament for inhibiting the formation of pathological neovascularization. The pathological neovascularization preferably includes choroidal neovascularization.

[0028] In the present invention, the most common treatment for age-related macular degeneration (AMD) or other neovascular retinal diseases is intravitreal injection of anti-vascular endothelial growth factor (VEGF) monoclonal antibodies. Therefore, anti-VEGF therapy is used as a positive control for the treatment of pathological neovascularization, and the results of spectral domain optical coherence tomography (SD-OCT) and (B) fluorescein angiography show that thrombospondin 1 (THBS-1) shows more obvious efficacy than anti-VEGF therapy in inhibiting pathological angiogenesis.

[0029] The present invention provides the use of thrombospondin 1 in the preparation of a medicament for preventing and / or treating retinal damage.

[0030] In the present invention, the retinal damage preferably includes retinal pigment epithelial cell damage. As a bridge between systemic blood circulation and neural retina, retinal pigment epithelial cells are responsible for maintaining the balance between subretinal space inflammatory response and angiogenic response through material exchange between the retina and adjacent tissues, and are prone to retinal inflammation and angiogenic response when the RPE is damaged by adverse stimuli such as blue light. The results of evaluating retinal visual function using a double method of morphological and electroretinogram analysis show that THBS-1 has a significant protective effect on blue light irradiated retinal visual function. The present invention finds that the mechanism of THBS-1 is induced by its binding to the receptor CD36, and therefore, a functional peptide segment selected for its CD36 binding can also achieve the same effect.

[0031] The use of thrombospondin 1 in the preparation of a medicament for preventing and / or treating retinal damage provided by the present invention will be described in detail below with reference to the examples, but they should not be understood as limiting the scope of protection of the present invention.

[0032] Example 1

[0033] Thrombospondin 1 (THBS-1) can effectively inhibit blue light-induced retinal inflammatory response

[0034] 1. Cell culture

[0035] ARPE-19 cells (human retinal pigment epithelial cell line) were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. The cells were cultured in high glucose DMEM medium containing 10% FBS and 1% penicillin-streptomycin double antibody, and placed in a 37°C and 5% CO2 incubator for culture, and subcultured at a ratio of 1:3 after about 2-3 days.

[0036] 2. THBS-1 administration

[0037] In the in vitro experiment, 4 nM of recombinant human thrombospondin-1 (THBS-1) was added to the cells in serum-starved condition and incubated for at least 6 hours before blue light irradiation.

[0038] 3. Cell grouping description

[0039] THBS-1 administration treatment + blue light irradiation group: ARPE-19 cells were seeded in 96-well plates at a density of 8 x 10 3 Cells were seeded in 96-well plates at a density of 8 x 10

[0040] Blue light irradiation group: ARPE-19 cells cultured as described above were seeded in 96-well plates at a density of 8 x 10 3 Cells were seeded in 96-well plates at a density of 8 x 10

[0041] Control group: ARPE-19 cells cultured as described above were seeded in 96-well plates at a density of 8 x 10 3 Cells were seeded in 96-well plates at a density of 8 x 10

[0042] 3. CCK-8 experiment

[0043] The cell viability of the above three groups of ARPE-19 cells was evaluated using CCK-8 reagent. At each time point of treatment, 10 μL of CCK-8 reagent was added to each well, and the ARPE-19 cells were incubated with the CCK-8 reagent at 37°C for 4 hours. After incubation, the absorbance was measured at a wavelength of 450 nm using an EPOCH2 microplate reader. The absorbance value at 0h of each group was taken as 100%, and normalization was performed.

[0044] 4. Live / dead cell staining experiment

[0045] ARPE-19 cells (including the THBS-1 administration treatment + blue light irradiation group and the non-blue light irradiation group) were seeded in 24-well plates at a density of 4 x 10 4 Cells in the light irradiation group were treated with blue light irradiation for 24 hours. The live / dead cell staining kit (Invitrogen) was used to stain the ARPE-19 cells cultured in the 24-well plates. The live cell staining reagent calcein and the dead cell staining reagent PI solution were added to the culture medium and incubated with the cells for 15 minutes. Random fields were selected under a fluorescence microscope and photographed and recorded.

[0046] 5. RNA extraction, reverse transcription and real-time quantitative polymerase chain reaction (RT-qPCR)

[0047] 1) Extraction of total cellular RNA

[0048] Cells to be extracted RNA (including THBS-1 administration treatment + blue light irradiation group and blue light irradiation group) were gently washed with PBS, then 1 mL of TRIzol was added to each sample, and the mixture was thoroughly mixed with a pipette gun. After lysis on ice for 10 minutes, the lysed cell liquid was transferred to a clean 1.5 mL nuclease-free EP tube. 500 μL of chloroform was added to each EP tube, and the mixture was vortexed vigorously for 30 seconds on a vortex. After standing on ice for 5 minutes, the 4°C pre-cooled centrifuge was centrifuged at 12,000 rpm for 20 minutes. Carefully pipette 500 μL of supernatant into a new EP tube, being careful not to aspirate the lower components to avoid affecting the purity of the nucleic acid. Add an equal volume of pre-cooled isopropanol, mix well by inverting, and stand at -20°C for 2 hours. Then centrifuge at 12,000 rpm and 4°C for 15 minutes, then discard the supernatant and add 1 mL of 75% ice ethanol (DEPC water + anhydrous ethanol) to each tube. Wash the precipitate. Centrifuge at 12,000 rpm and 4°C for 15 minutes, discard the supernatant, and dry the liquid in the EP tube. Then add an appropriate amount of DEPC water to dissolve the precipitate. Use a NanoDrop spectrophotometer to detect OD 260 / OD 280 , to detect the extraction purity and nucleic acid concentration.

[0049] 2.) Reverse transcription

[0050] Takara's PrimeScriptTM Reverse Transcription Kit was used for reverse transcription of RNA. The total amount of RNA extracted from each sample was 1 μg, 4 μL of 5×Prime Script TM buffer was added, 1 μL of Prime Script TM enzyme, 1 μL of Random6mers, 1000 / RNA concentration of RNA was added, and DEPC water was added to make the system 20 μL. The reverse transcription PCR instrument cycle parameters were 37°C for 15 minutes, 85°C for 5 seconds, and the temperature was lowered to 4°C. The reverse-transcribed sample was stored at -20°C.

[0051] 3) qPCR reaction

[0052] The qPCR reaction system was 1 μL of 10-fold diluted cDNA, 5 μL of Power SYBR Green PCR MasterMix enzyme, 1 μL of 10-fold diluted target gene primer (see Table 1), and 3 μL of DEPC water. The qPCR cycle reaction conditions are shown in Table 2. The sample was added to a 384-well plate, the plate was sealed, and the 384-well plate was centrifuged at 2000 rpm for 2 minutes. The 384 plate was placed into the qPCR instrument, the reaction system was set to 10 μl, and the amplification was 40 cycles. Finally, the results were analyzed by △△CT.

[0053] Table 1 qPCR primer information

[0054]

[0055]

[0056] Table 2 qPCR reaction conditions

[0057]

[0058] 6. Western blot experiment

[0059] The lysate containing RIPA, 100x protease inhibitor and 100x phosphatase inhibitor was prepared, and the cells were washed with PBS once. For experiments detecting VEGFR2 phosphorylation and downstream pathway phosphorylation, 20 ng / mL VEGF was added to the cells for incubation for 30 minutes before protein extraction. After adding the prepared protein lysate, it was lysed on ice for half an hour. The protein lysate was collected in a clean 1.5 mL EP tube, and ultrasonicated on ice for 6 times, each time for 10 seconds and then paused for 10 seconds. The pre-cooled 4°C centrifuge was used to centrifuge at 12000 rpm for 20 min. The supernatant was taken to a new 1.5 mL EP tube, and prepared for protein quantification analysis. The BCA protein quantification kit (Thermo Fisher) was taken, and the BCA quantification reagent was prepared according to the ratio of A liquid:B liquid = 50:1. The protein standard was set to different concentration gradients to draw a standard curve. Different amounts of protein standard and the same volume of protein sample were added to each well of the 96-well plate. 150 μL of BCA quantification reagent was added to each well, and incubated at 37°C for 30 minutes. The absorbance was measured at 539 nm wavelength by using the enzyme-labeled instrument. The data was analyzed by Excel table and the standard curve of protein concentration was drawn, taking absorbance and concentration as coordinate axes, fitting the formula, and substituting the measured absorbance into the formula to obtain the protein sample concentration to determine the final protein loading amount.

[0060] SDS-PAGE gel was prepared before running the gel. The sample loading amount was determined according to the quantification result. The running voltage was 80V for 30 minutes and 120V for 40 minutes. After the protein bands were separated, the SDS-PAGE gel electrophoresis was stopped and the protein was transferred. The PVDF membrane was activated in methanol before being transferred. The transfer solution was pre-cooled in ice. The transfer was performed at 4°C with a constant current of 200-300mA for a certain time, which was determined according to the protein molecular weight. After the transfer, the PVDF membrane was blocked with 5% BSA or 5% skim milk at room temperature for 1 hour. Then the PVDF membrane was incubated with specific antibodies at 4°C overnight. After the incubation with the primary antibody, the membrane was washed with TBST for three times, each for 10 minutes. Then the membrane was incubated with the secondary antibody with HRP binding site corresponding to the species of the primary antibody at room temperature for 1 hour. After the incubation with the secondary antibody, the membrane was washed with TBST for three times, each for 10 minutes. The HRP developing solution was prepared and the membrane was scanned.

[0061] 7. Immunocytochemistry experiment

[0062] ARPE-19 cells (including THBS-1 administration + blue light irradiation group and blue light irradiation group) were seeded on cell-crawling slides in 12-well plates or 24-well plates. After blue light treatment, the cells were rinsed with PBS once, and then fixed with 4% paraformaldehyde for 30 minutes. After rinsing with PBS for three times, the cells were blocked with blocking solution containing 0.3% Triton X-100 and 10% goat serum for 1 hour. After blocking, different primary antibodies were used to incubate the cells at 4°C overnight. After the incubation with the primary antibody, the cells were rinsed with PBS for three times, each for 5 minutes. The secondary antibody with fluorescent probe was used to incubate the cells for 1 hour in the dark. Then the cell nuclei were counterstained with DAPI for 15 minutes. The cell-crawling slides were inverted on glass slides, and three random fields were randomly selected for imaging the cells using a fluorescence microscope.

[0063] Experimental results

[0064] 1. Blue light irradiation induces abnormal levels of inflammatory factors in ARPE-19 cells

[0065] Inflammation is considered one of the important pathological processes of retinal degenerative diseases such as age-related macular degeneration (AMD), and the inflammatory response in RPE greatly promotes the development of retinal degenerative diseases. Compared with the control group (ARPE-19 cells without blue light irradiation), the gene ( Figure 2 A) and protein ( Figure 2 B) expression levels of pro-inflammatory cytokines related to retinal inflammation (including MCP-1, TNF-α, IL-1β, IL-6 and IL-8) were significantly increased, while the expression of immunosuppressive cytokine TGF-β1 was decreased, indicating that blue light induced an inflammatory response in RPE cells.

[0066] 2. Blue light induces inflammatory activity of ARPE-19 cells through activation of NF-κΒ pathway

[0067] By detecting the core transcription factors of multiple inflammation-related signaling pathways, including IKBa (NF-κΒ signaling pathway), IRAK-1 (Toll-like receptor 4 signaling pathway), c-Jun (c-Jun / AP1 signaling pathway), STAT3 (Jak / STAT3 signaling pathway), and p38 (p38 / MAPK signaling pathway), among the core transcription factors regulating these signaling pathways, IRAK-1, c-Jun, STAT3, and p38 did not show significant phosphorylation activation ( Figure 2 A), while IKBa showed significant phosphorylation activation in blue light-exposed ARPE-19 cells, in addition, the protein expression of NF-κΒ p65 (downstream of IKBa) was down-regulated in the cytoplasm, while the protein expression in the nucleus was increased ( Figure 3 B), and the nuclear translocation of NF-κΒ p65 was significantly increased in blue light-exposed ARPE-19 cells by immunocytochemistry experiments ( Figure 3 C), which suggests that the up-regulation of pro-inflammatory cytokines is mainly caused by the NF-κΒ signaling pathway mediated by blue light to cause RPE inflammation. After P-IKBa inhibitor BAY 11-7082 treatment, the protein level of pro-inflammatory cytokines was significantly reversed ( Figure 3 D), further confirming the activation of the NF-κΒ inflammatory pathway in blue light-induced inflammation. The data show that blue light activates the NF-κΒ inflammatory pathway by stimulating the phosphorylation of IKBa in RPE cells and further increasing the nuclear translocation of NF-κΒ p65, ultimately activating the transcription of multiple downstream pro-inflammatory cytokines.

[0068] 3. THBS-1 inhibits blue light-induced retinal inflammation by inhibiting the NF-κΒ pathway

[0069] Based on the previous research, it has been known that blue light mediates the abnormal expression of inflammatory factors in the retina through the activation of the NF-κΒ signaling pathway, in order to explore whether THBS-1 plays a role in blue light-induced inflammation, the effect of THBS-1 on the expression level of NF-κΒ signaling pathway-related proteins in blue light-irradiated ARPE-19 cells was first explored. Western blot results showed that after THBS-1 treatment, the phosphorylation activation of IKBa and the nuclear translocation of NF-κΒ p65 mediated by blue light were inhibited ( Figure 3 A), indicating that THBS-1 can inhibit the NF-κΒ inflammatory signaling pathway activated by blue light. qPCR ( Figure 3 B) and Western blot ( Figure 3C) Results showed that blue light can induce the increase of the levels of inflammatory cytokines downstream of NF-κΒ, including MCP-1, TNF-a, IL-1β, IL-6 and IL-8 in ARPE-19 cells, while the expression level of the immunosuppressive cytokine TGF-β1 was decreased after blue light irradiation, and THBS-1 can significantly reduce the expression of pro-inflammatory factors induced by blue light and increase the expression of TGF-β1. Similarly, the number of MCP-1 and CD68 (a marker of inflammatory monocytes) positive cells in the mouse retina induced by blue light was also significantly reduced by THBS-1 treatment Figure 4 D Figure 4 E), which indicates that THBS-1 can significantly inhibit the inflammatory response and the subsequent recruitment of macrophages / monocytes into the immune-privileged subretinal area after blue light exposure. In summary, the results indicate that THBS-1 plays a key role in inhibiting blue light-induced retinal inflammation by inhibiting the NF-κΒ signaling pathway Figure 4 F).

[0070] Example 2

[0071] Platelet reaction protein 1 (THBS-1) can effectively inhibit pathological neovascularization

[0072] I. hMEC-1 cell level experiment

[0073] 1. Cell culture

[0074] hMEC-1 cells (human microvascular endothelial cell line) were purchased from the American ATCC cell bank. The cells were cultured in ECM (endothelial cell medium) containing 5% FBS, 1% penicillin-streptomycin double antibody and 1% endothelial cell growth supplement (ECGS) in a 37°C, 5% CO2 incubator, and the medium was changed every day, and the cells were passaged at a ratio of 1:2 about 2-3 days later.

[0075] 2. THBS-1 administration treatment

[0076] When detecting the expression of phosphorylated proteins in hMEC-1 cells, the cells were pretreated with 4 μM THBS-1 solution for 6 h in a serum-starved state, and 20 ng / mL VEGF was added to the culture medium without ECGS for 30 min.

[0077] 3. Cell grouping

[0078] THBS-1 administration treatment + VEGF treatment group: cells were seeded in 96-well plates at a density of 8 x 10 3 Cells were seeded in 96-well plates at a density of 8 x 10

[0079] VEGF treatment group: cells were seeded at a density of 8 x 10 3 Cell seeding at a density of 8 x 10

[0080] Control group: cells were seeded at a density of 8 x 10 3 Cell seeding at a density of 8 x 10

[0081] 4. EdU cell proliferation assay

[0082] When the cell confluence reached 60-80%, the cells were starved in low serum medium (1% FBS) for 12 hours to keep the same proliferation cycle before subsequent treatment. After the corresponding time point, EdU assay was performed by using EdU kit (RiboBio, China) to evaluate the proliferation activity of cells. The specific steps are as follows: prepare glycine solution and PBS permeabilizing solution containing 0.5% Triton X-100, dilute EdU reagent with cell culture solution, prepare EdU medium, incubate cells with EdU medium for 2 hours, and rinse with PBS for 1-2 times. Then, fix the cells with 4% paraformaldehyde for 30 minutes, add 2 mg / mL glycine solution per well, incubate for 5 minutes, rinse with PBS, then add 0.5% Triton X-100 PBS per well and incubate for 10 minutes, and then rinse with PBS once. Then, re-stain with Apollo dye for 30 minutes, and then add permeabilizing agent and incubate for 10 minutes. After that, incubate the cells with DAPI for 30 minutes. Then, randomly select fields of view using a fluorescence microscope (Nikon) to take pictures. Quantify using ImageJ software, and determine the cell proliferation rate as proliferation rate = (EdU positive cells / DAPI stained cells) x 100%.

[0083] 5. Cell scratch assay

[0084] When the confluence of hMEC-1 cells reached 100%, a uniform scratch was made on the monolayer cells using a 200 μL pipette tip, and the scraped cell fragments were washed away with PBS. The cells were again incubated with fresh medium containing 1% FBS to put the cells in a serum-starved state to exclude the proliferation effect. Then, the scratch image at 0 hours was taken and recorded under a light microscope. After 48 hours, the image of the same scratch position recorded at 0 hours was taken and recorded. Finally, the scratch confluence area was quantitatively calculated by ImageJ to evaluate the lateral migration ability of the cells.

[0085] 6. Transwell assay

[0086] The transwell chamber with a pore size of 8.0 μm was used to detect the longitudinal migration ability of hMEC-1 cells. 200 μL of cells (4 x 104 The cells were seeded into 24-well plates transwell chambers, and 4nM THBS-1 was added to the upper chamber of the THBS-1 treatment group, while 800 μL of ECM medium with or without 20 ng / ml VEGF was added to the lower chamber. After 24 hours of culture, the transwell chambers were removed, carefully rinsed with PBS, and fixed with 4% paraformaldehyde for 30 minutes. Then, the cells that did not migrate were carefully removed from the upper surface of the transwell with a wet cotton swab. Next, the migrated cells were stained with 1% crystal violet solution for 15 minutes, and after staining, the chambers were air-dried, and images were collected under a microscope. The number of migrated cells was quantified using ImageJ software.

[0087] 7. Cell tube formation experiment

[0088] The Matrix gel (Corning, USA) stored at -20°C was thawed at 4°C, placed on ice, and the 24-well plate was pre-cooled on ice, and the 200 μL pipette was pre-cooled at -20°C. The pre-cooled pipette was used to draw 200 μL of Matrix gel and evenly spread it on the 24-well plate and incubated in a 37°C incubator for 1 hour to solidify the Matrix gel into a solid state. Then, 4 x 10 4 The hMEC-1 cells were seeded into the 24-well plate coated with Matrix gel at a density of 4 x 10

[0089] Experimental results

[0090] 1. Angiogenic activity of hMEC-1 cells induced by ARPE-19 cell conditioned medium after blue light irradiation

[0091] Under physiological conditions, the subretinal region is an avascular region. Considering that pathological neovascularization in the subretinal region is another key pathological process in addition to inflammation in retinal degenerative diseases such as AMD, it was next investigated whether blue light would cause abnormal activation of angiogenic activity. Co-culturing of conditioned medium from ARPE-19 cells exposed to blue light (BL-CM) with hMEC-1 cells (human microvascular endothelial cell line) mimicked the regulatory role of RPE cells on the angiogenic balance. The proliferation, migration and tube formation capacity of vascular endothelial cells are important indicators for evaluating the angiogenic activity of vascular endothelial cells, and here, by treating hMEC-1 cells with BL-CM or medium from untreated RPE cells, the activation of their angiogenic response was evaluated by assessing their proliferation, migration and tube formation capacity. The results of the EdU experiment showed that the proportion of EdU-positive cells significantly increased after treatment with BL-CM, indicating an increase in the proliferative activity of hMEC-1 cells Figure 4 A). In addition, the transverse and longitudinal migration capacity of BL-CM-treated hMEC-1 cells was significantly enhanced relative to control cells, as shown by scratch experiments Figure 5 B) and Transwell experiments Figure 5 C), respectively. Tube formation experiments are one of the most important parameters for detecting angiogenic responses, and the results of the tube formation experiment analysis showed that the number of branches, nodules and lumens of tubes formed by BL-CM-treated cells was significantly increased compared to control cells, demonstrating that the tube formation capacity of hMEC-1 cells in the BL-CM group was significantly enhanced Figure 5 D). These data indicate that by treating with BL-CM, the angiogenic response of hMEC-1 cells was significantly activated, mimicking the damage to the regulatory role of RPE cells on the angiogenic balance caused by external blue light exposure.

[0092] 2. THBS-1 inhibits VEGF-mediated angiogenic responses in hMEC-1 cells

[0093] Based on the above mechanisms, the role of THBS-1 in regulating angiogenic responses in hMEC-1 cells in vitro was next investigated. The results of the EdU experiment showed that THBS-1 significantly inhibited VEGF-induced endothelial cell proliferation Figure 5 A- Figure 5 B). By scratch experiments Figure 5 C- Figure 5 D) and Transwell experiments Figure 5 E- Figure 5 F) showed that VEGF significantly promoted the transverse and longitudinal migration of hMEC-1 cells compared to untreated control cells, and this promotion was significantly inhibited by THBS-1. In addition, the effect of THBS-1 on the tube formation capacity of hMEC-1 was evaluated by tube formation experimentsFigure 6 G), the quantification of the number of nodes, branches and lumens in the tube formation results showed that THBS-1 significantly inhibited the tube formation ability of VEGF-induced hMEC-1 Figure 6 H Figure 6 J). These results showed the effective inhibition of THBS-1 on angiogenesis in vitro.

[0094] II. Animal model level experiments

[0095] 1. Introduction of animal materials

[0096] Eight-week-old male C57BL / 6J mice were used as experimental objects and placed in a specific pathogen-free (SPF) environment with a 12-hour day-night cycle. All experiments were performed in accordance with the animal experiment standards of the Association for Research in Vision and Ophthalmology (ARVO) and were approved by the Animal Ethics Committee of the Ninth People's Hospital of Shanghai Jiaotong University School of Medicine.

[0097] 2. Construction of retinal blue light damage model

[0098] All mice were divided into two groups: (1) mice without any treatment (control group); (2) blue light exposure group (BL group), mice in the blue light exposure group were exposed to LED blue light for 7 days, followed by a 12-hour blue light-dark cycle (light cycle ratio: 12h / 12h). Pupil dilation was performed once every 3 hours with eye drops containing tropicamide (0.5%) and phenylephrine (0.5%). After one week, some mice were sacrificed and the eyeballs were collected for subsequent analysis.

[0099] 3. Grouping of retinal blue light damage model

[0100] All mice were divided into three groups: (1) mice without any treatment (control group) (2) mice injected with PBS in the vitreous cavity one day before blue light exposure (BL group); (3) mice injected with 10 μg / mL THBS-1 in the vitreous cavity one day before blue light exposure (BL+THBS-1 group). One day after administration, mice in the BL and BL+THBS-1 groups were exposed to LED blue light for 7 days, followed by a 12-hour blue light-dark cycle. Pupil dilation was performed once every 3 hours with eye drops containing tropicamide (0.5%) and phenylephrine (0.5%). After one week, all mice were sacrificed and the eyeballs were collected for subsequent analysis.

[0101] 4. Immunohistochemical experiments

[0102] Mouse eyeballs were removed and immediately stored in FAS eyeball fixative (Servicebio). Paraffin-embedded tissue blocks were cut into a thickness of about 5 pm and stained with an antibody against VEGF. The primary antibody was bound with a fluorescent secondary antibody (Alexa Fluor 488 / 594, Invitrogen, 1:500). Photographic shooting was performed under a microscope, and all images were randomly taken from at least three areas of each section. The ONL thickness of the entire retina was quantified using the ImageJ program.

[0103] 5. Laser-induced CNV model and drug treatment

[0104] After the mice were deeply anesthetized, the pupils of the mice were diffused with mydriatic agents, and self-made mouse corneal contact lenses were placed on the corneas of the mice to facilitate magnification and observation of the fundus. The parameters of the argon laser photocoagulation instrument were set as follows: wavelength of 532 nm, power of 575 mw, exposure time of 50 ms, and spot size of 50 pm. The mice were placed on the slit lamp platform, and the fundus of the mice was observed through the slit lamp. Laser photocoagulation was performed, and 4 to 5 laser spots were fired around the optic disc to avoid blood vessels, and the distance from the optic disc should not be too far to avoid the whole eye fundus from being unable to be photographed. Seven days after photocoagulation, new blood vessels gradually formed, at which time all the mice were randomly divided into 3 groups, namely: (1) intravitreal injection of 3 pL PBS (PBS group) after laser treatment for 1 week; (2) intravitreal injection of 3 pL 10 pg / mL THBS-1 (THBS-1 group) after laser treatment for 1 week; (3) intravitreal injection of 3 pL 10 mg / mL anti-VEGF (Lucentis, Novartis) (anti-VEGF group).

[0105] 6. Retinal optical coherence tomography (SD-OCT) and fluorescein angiography (FA)

[0106] SD-OCT and FA detection were performed at 7 days, 14 days, and 21 days after CNV modeling. Seven days after laser photocoagulation, choroidal neovascularization was basically formed, at which time PBS (as a negative control group), THBS-1, or anti-VEGF (as a positive control group) was injected intravitreally according to the grouping. After the mice were anesthetized with chloral hydrate, tropicamide phenylephrine eye drops were used to diffuse the pupils. Then the mice were placed on the platform of the SD-OCT system (Heidelberg Engineering, Germany), and the results of SD-OCT were obtained. The eye fundus fluorescein angiography (FA) was imaged using the SD-OCT system (Heidelberg Engineering, Germany) after intraperitoneal injection of 50 pL of 10% fluorescein sodium (Alcon) solution. The fluorescence intensity was quantified using ImageJ software to measure the level of fluorescein leakage.

[0107] 7. Statistical analysis

[0108] All current statistics are from at least three independent repeated trials and are presented as mean ± standard deviation. Data between two independent groups were analyzed using Student's t-test. For statistical analysis among three or more experimental groups, one-way ANOVA was used, followed by LSD post-hoc t-tests. In time-related data analysis, statistical analysis was performed at each time point for each experimental group. P < 0.05 was considered statistically significant. * and ** are used to indicate P < 0.05 and P < 0.01, respectively, in the figures.

[0109] Experimental results

[0110] 1. THBS-1 inhibits choroidal angiogenesis in C57BL / 6 mice.

[0111] Based on the inhibitory activity of THBS-1 on in vitro angiogenesis, the potential of THBS-1 in in vivo anti-angiogenesis in animal experiments was further investigated. In C57BL / 6 mice exposed to blue light, high expression of VEGF was observed around the retinal retinopathy (RPE). VEGF is the most important factor promoting angiogenesis in retinal neovascularization, and THBS-1 treatment significantly reduced the level of VEGF in the subretinal space. Figure 6 A). To better evaluate the anti-angiogenic ability of THBS-1 in the retina, a mouse model of laser-induced choroidal neovascularization (CNV) was established. This model is widely used as the gold standard for CNV research in neovascular retinal degenerative diseases. Anti-VEGF therapy is currently the main method for treating neovascular retinal degenerative diseases such as AMD. Therefore, the anti-VEGF treatment group was used as the positive control group, and the PBS treatment group was used as the negative control group. After successfully establishing the neovascularization model (7 days after laser injury), 3 μL of PBS, THBS-1, and anti-VEGF were injected into the vitreous cavity of mice in different groups according to their respective groups. SD-OCT scans of the laser injury images showed that, compared with the PBS treatment group, the THBS-1 treatment group and the anti-VEGF treatment group ( Figure 7 B) Reduced thickness of laser-induced CNV lesions (as indicated by the red arrows). As shown in fluorescein angiography (FA), pathological neovascularization of the choroid in PBS-treated mice exhibited continuous leakage. Compared to the pre-administration modeled area, THBS-1 treatment and anti-VEGF treatment significantly reduced the neovascular leakage area by approximately 85% and 70%, respectively, on day 14 post-administration. Figure 7 C~ Figure 8D). Notably, THBS-1 was comparable to anti-VEGF treatment, and even superior to anti-VEGF antibody in reducing neovascular leakage. Taken together, these data suggest that THBS-1 effectively inhibits angiogenesis in vitro and in vivo by inhibiting VEGF-mediated VEGFR2 signaling pathway through CD36.

[0112] Example 3

[0113] Platelet Reactin 1 (THBS-1) can significantly protect the retina from blue light-induced retinal damage

[0114] I. ARPE-19 cell level experiment

[0115] 1. Cell culture

[0116] ARPE-19 cells (human retinal pigment epithelial cell line) were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. The cells were cultured in high glucose DMEM medium containing 10% FBS and 1% penicillin-streptomycin double antibody, and placed in a 37°C and 5% CO2 incubator for culture, and about 2-3 days later, the cells were subcultured at a ratio of 1:3.

[0117] 2. THBS-1 administration treatment

[0118] In the in vitro experiment, 4nM recombinant human platelet reaction protein-1 (THBS-1) was added to the cells in a serum-starved state, and cultured for at least 6 hours before blue light irradiation.

[0119] 3. Cell grouping

[0120] THBS-1 administration treatment + blue light irradiation group: 8x10 3 cells / well were seeded in a 96-well plate, and after the cells were treated according to the method of item 2 above, blue light irradiation was performed for 24 hours (the wavelength of blue light was 426nm) after 6 hours of culture.

[0121] Blue light irradiation group: the above cultured ARPE-19 cells were seeded in a 96-well plate at a density of 8x10 3 cells / well, and blue light irradiation was performed for 24 hours.

[0122] Control group: the above cultured ARPE-19 cells were seeded in a 96-well plate at a density of 8x10 3 cells / well, and no blue light irradiation was performed.

[0123] 4. RNA extraction, reverse transcription and real-time quantitative polymerase chain reaction (RT-qPCR)

[0124] The expression level of the genes of tight junction proteins Occludin and ZO-1 in ARPE-19 cells was detected according to the method of Example 1, and the amplification primers of the genes of Occludin and ZO-1 are shown in Table 3. The qPCR cycle reaction conditions are shown in Table 2 above. The sample was added to a 384-well plate, sealed, and the 384-well plate was centrifuged at 2000 rpm for 2 minutes. The 384-well plate was placed in a qPCR reaction instrument, the reaction system was set to 10 μl, and amplification was performed for 40 cycles. Finally, the results were analyzed by △△CT.

[0125] Table 3 qPCR primer information

[0126]

[0127] 4. Immunocytochemical analysis

[0128] The cells were subjected to immunocytochemical analysis according to the method described in Example 1.

[0129] 5. EdU cell proliferation detection

[0130] When the cell fusion reached 60%-80%, the cells were starved in low serum medium (1% FBS) for 12 hours to keep the cells in the same proliferation cycle before subsequent treatment. After the corresponding time point, EdU analysis was performed by using an EdU kit (RiboBio, China) to evaluate the proliferation activity of the cells. The specific steps are as follows: prepare a glycine solution, and a PBS permeating solution containing 0.5% Triton X-100, dilute the EdU reagent with cell culture solution, prepare EdU medium, incubate the cells with EdU medium for 2 hours, and rinse with PBS for 1-2 times. Then, fix the cells with 4% paraformaldehyde for 30 minutes, add 2 mg / mL glycine solution per well, incubate for 5 minutes, rinse with PBS, then add 0.5% Triton X-100 in PBS per well and incubate for 10 minutes, followed by PBS rinsing once. Then, re-stain with Apollo dye for 30 minutes, and then add the permeating agent and wash for 10 minutes. After that, incubate the cells with DAPI for 30 minutes. Then, randomly select fields of view using a fluorescence microscope (Nikon) to take pictures. Quantify using ImageJ software, and determine the cell proliferation rate with the proliferation rate = (EdU positive cells / DAPI stained cells) x 100%.

[0131] 6. Live / dead cell staining experiment

[0132] Add 4 x 10 4The density of cells will be ARPE-19 cells (including THBS-1 administration treatment + blue light irradiation group and blue light irradiation group) seeded in 24-well plates, and the cells of the light irradiation experimental group will be treated with blue light irradiation for 24 hours. The cell viability of ARPE-19 cells after blue light irradiation is evaluated by using the live / dead cell staining experiment, and the ARPE-19 cells cultured in the 24-well culture plate are stained by using the live / dead cell staining kit (Invitrogen). The live cell staining reagent calcein and the dead cell staining reagent PI solution are added to the culture solution to incubate the cells for 15 minutes, and the field of view is randomly selected by using a fluorescence microscope to take pictures and record.

[0133] 7. Western blot experiment

[0134] The expression level of tight junction proteins (Occludin and ZO-1) is detected according to the Western blot experiment method described in Example 1.

[0135] II. Animal model level experiment

[0136] 1. Construction of retinal blue light damage model

[0137] All mice are divided into two groups: (1) mice without any treatment (control group); (2) blue light irradiation group (BL group), and the mice in the blue light irradiation group are exposed to LED blue light for 7 days, and then kept in a 12-hour blue light-dark cycle (light-dark cycle ratio 12h:12h). Pupil dilation is performed once every 3 hours by using eye drops containing tropicamide (0.5%) and phenylephrine (0.5%). After one week, part of the mice are sacrificed, and the eyeballs are collected for subsequent analysis.

[0138] 2. Grouping of retinal blue light damage model

[0139] All mice are divided into three groups: (1) mice without any treatment (control group) (2) mice injected with PBS in the vitreous cavity one day before blue light irradiation (BL group); (3) mice injected with 10 μg / mL THBS-1 in the vitreous cavity one day before blue light irradiation (BL+THBS-1 group). One day after administration, the mice in the BL group and the BL+THBS-1 group are exposed to LED blue light for 7 days, and then kept in a 12-hour blue light-dark cycle. Pupil dilation is performed once every 3 hours by using eye drops containing tropicamide (0.5%) and phenylephrine (0.5%). After one week, they are used for subsequent analysis.

[0140] 3. Electroretinogram (ERG)

[0141] Mice were dark-adapted for 24 hours in a dark environment before ERG analysis. The mice were placed under dim red light for anesthesia and pupil dilation. Throughout the experiment, the mice were placed on a warm pad to exclude interfering stimuli. Grounding electrodes were attached to the tail and reference electrodes were attached to the forehead. Two gold ring electrodes were placed on the cornea of the mice and the responses of the retina to light stimuli were recorded using an Espion E3 instrument (Diagnosys, Boxborough, MA). The dark ERG was evoked with a first 10 cd*s / m 2 2 flash of light. The ERG was performed at a flash intensity of 30 cd*s / m 2 2 and followed by 5 minutes of bright adaptation. The a-wave amplitude was measured as the voltage from baseline to the lowest negative voltage and the b-wave amplitude was measured from the trough of the a-wave to the peak of the b-wave.

[0142] Results

[0143] 1. Blue light irradiation affects tight junctions of ARPE-19 cells

[0144] The tight junction function of retinal pigment epithelial cells (RPE) is the guarantee of RPE as the blood-retinal barrier, which is essential for maintaining retinal homeostasis. By detecting the gene and protein expression levels of RPE cell tight junction proteins Occludin and ZO-1, qPCR results showed that the mRNA expression levels of Occludin and ZO-1 decreased after short-wave blue light irradiation for 24 hours ( Figure 8 A), and immunocytochemistry results showed that in normal RPE cells, Occludin and ZO-1 were expressed completely at the junction between cells, while after short-wave blue light irradiation for 24 hours, the expression of Occludin and ZO-1 decreased significantly ( Figure 8 B), further supporting that blue light can induce damage to RPE cell tight junction proteins, leading to damage to the tight junction function of RPE cells, simulating the retinal damage changes of retinal degenerative diseases.

[0145] 2. Protective effect of THBS-1 on blue light damage of retina (RPE)

[0146] Based on the anti-inflammatory and anti-angiogenic mechanisms of THBS-1, it was further explored whether THBS-1 played a certain role in protecting the retina (RPE) from blue light-induced damage. According to the EdU experiment ( ​ A), live / dead cell staining ( ​B) The results showed that THBS-1 significantly restored the proliferative capacity and cell viability of ARPE-19 cells reduced by blue light irradiation. In order to characterize the effectiveness of THBS-1 treatment from a molecular point of view, the protein expression levels of RPE-specific tight junction proteins Occludin and ZO-1 were analyzed by immunocytochemistry, THBS-1 can significantly rescue the reduced expression of tight junction proteins in ARPE-19 cells exposed to blue light ​ C). These results show that THBS-1 can protect RPE cells from blue light-induced damage in vitro.

[0147] In summary, THBS-1 can achieve the dual effects of anti-inflammation and anti-angiogenesis by inhibiting the NF-κB and VEGFR2 signaling pathways, inhibit blue light-induced retinal inflammation and neovascularization, alleviate the disorder of retinal structure and function, and show excellent protective effect of THBS-1 on retinal degenerative diseases retinal damage.

[0148] The above only describes the preferred embodiments of the present application, and it should be pointed out that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. Use of Thrombospondin 1 in the preparation of a medicament for preventing retinal damage; the retinal damage being induced by blue light; the retinal damage including retinal pigment epithelium cell damage; the amino acid sequence of the Thrombospondin 1 being as set forth in SEQ ID NO: 1 of the Sequence Listing.

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