Application of VEGF-B protein and VEGF-B protein active peptide fragment in preparing medicine for preventing and / or treating acute anterior uveitis

Abstract

The present invention discloses the use of VEGF-B protein and VEGF-B protein active peptides in the preparation of drugs for preventing and / or treating acute anterior uveitis. The VEGF-B protein and VEGF-B protein active peptides provided by the present invention have good drug safety and anti-acute anterior uveitis effects, can reduce the number of ocular inflammatory cells and inhibit the expression of inflammatory factors, and can inhibit macrophage M1 polarization, providing new ideas for the prevention and treatment of eye diseases, and have very broad application prospects in the medical field.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the use of VEGF-B protein and VEGF-B protein active peptide segments in the preparation of drugs for preventing and / or treating acute anterior uveitis. Background Art

[0002] Vascular endothelial growth factor VEGF-B (Vascular endothelial growth factor B) is a member of the VEGF family. It mainly binds to vascular endothelial growth factor receptor-1 (VEGFR-1) and neuropilin 1 (NRP-1) and is expressed in most tissues and organs. When VEGF-B specifically binds to VEGFR-1, vascular endothelial cells can respond to VEGF-B by increasing the expression and activity of plasminogen activator inhibitor 1 (PAI-1) and urokinase-type plasminogen activator (uPA); and NRP-1, as a receptor for brain degeneration protein / signalin, has the function of guiding neuronal growth cones and can selectively bind to VEGF-B to exert its effect.

[0003] Uveitis is a type of intraocular inflammatory disease that mainly affects the pigmented membranes of the eye, such as the iris, ciliary body, and choroid. Severe cases can even lead to blindness, accounting for 10% to 15% of blind patients worldwide. In China, about 4% to 10% of blind patients are blinded by uveitis. Acute anterior uveitis (AAU) is the most common type of uveitis, with an incidence of about 28% to 61%. The clinical manifestations of AAU include ciliary congestion, obvious anterior chamber flare, sudden eye pain, red eyes, photophobia, and blurred vision. In severe cases, posterior synechiae of the iris may form or worsen, complicated by glaucoma, resulting in irreversible damage or loss of vision. Drugs used clinically to treat anterior uveitis include: 1) mydriasis and ciliary muscle paralysis, which can only be used to relieve clinical symptoms such as pain and photophobia; 2) non-steroidal anti-inflammatory drugs, which are only effective for certain non-necrotizing anterior scleral inflammation, and systemic use will increase the risk of gastrointestinal ulcers; 3) steroids, the main drugs for the treatment of uveitis, are divided into local and systemic administration, with serious side effects, ocular side effects include cataracts, increased intraocular pressure and glaucoma, systemic side effects include diabetes, hypertension, peptic ulcers, insomnia, osteoporosis, etc.; 4) immunomodulatory therapy, such as anti-TNF-α, anti-IL-6, anti-IL-1β therapy, etc., can achieve better results when combined with traditional steroids or cellular immunosuppressants, but infection caused by immunosuppression is the main side effect. Since the drugs currently used clinically to treat acute anterior uveitis can only control inflammation to a certain extent and have serious side effects, which will further lead to vision loss, safe and effective target drugs for the prevention and treatment of acute anterior uveitis are urgently needed. Summary of the invention

[0004] The purpose of the present invention is to provide the use of VEGF-B protein or VEGF-B protein active peptide segment in the preparation of a drug for preventing and / or treating acute anterior uveitis.

[0005] According to one aspect of the present invention, the use of VEGF-B protein or VEGF-B protein active peptides in the preparation of a drug for preventing and / or treating acute anterior uveitis is provided. The VEGF-B protein and VEGF-B protein active peptides provided by the present invention have good anti-inflammatory effects, can reduce the number of ocular inflammatory cells and inhibit the expression of inflammatory factors, thereby playing a role in treating acute anterior uveitis, providing a new idea for the prevention and treatment of eye diseases, and having a very broad application prospect in the medical field.

[0006] Preferably, the drug for preventing and / or treating acute anterior uveitis is a drug for reducing ocular inflammatory cells.

[0007] Preferably, the inflammatory cells include CD45 positive cells (CD45+ cells).

[0008] Preferably, the active peptide segment of VEGF-B protein includes at least one of Peptide 1, Peptide 2, Peptide 3, Peptide 4 and Peptide 5; the amino acid sequence of Peptide 1 is shown as SEQ ID NO.1, the amino acid sequence of Peptide 2 is shown as SEQ ID NO.2, the amino acid sequence of Peptide 3 is shown as SEQ ID NO.3, the amino acid sequence of Peptide 4 is shown as SEQ ID NO.4, and the amino acid sequence of Peptide 5 is shown as SEQ ID NO.5.

[0009] Preferably, the active peptide segment of VEGF-B protein includes Peptide 5. Among the active peptide segments of VEGF-B protein, Peptide 5 has a stronger anti-inflammatory effect.

[0010] According to another aspect of the present invention, there is provided use of VEGF-B protein or an active peptide segment of VEGF-B protein in the preparation of a drug for inhibiting macrophage M1 polarization.

[0011] Preferably, the drug for inhibiting macrophage M1 polarization is a drug for reducing the expression of inflammatory factors.

[0012] Preferably, the inflammatory factors include at least one of TNF-α, IL-6, and IL-1β.

[0013] Preferably, the active peptide segment of VEGF-B protein includes at least one of Peptide 1, Peptide 2, Peptide 3, Peptide 4 and Peptide 5; the amino acid sequence of Peptide 1 is shown as SEQ ID NO.1, the amino acid sequence of Peptide 2 is shown as SEQ ID NO.2, the amino acid sequence of Peptide 3 is shown as SEQ ID NO.3, the amino acid sequence of Peptide 4 is shown as SEQ ID NO.4, and the amino acid sequence of Peptide 5 is shown as SEQ ID NO.5.

[0014] Preferably, the active peptide segment of VEGF-B protein includes Peptide 5. Among the active peptide segments of VEGF-B protein, Peptide 5 has a stronger anti-inflammatory effect.

[0015] Preferably, the VEGF-B protein comprises VEGF-B 167 .

[0016] According to another aspect of the present invention, a medicine is provided, which comprises VEGF-B protein and / or active peptide fragments of VEGF-B protein as active ingredients.

[0017] The present invention has the following beneficial effects:

[0018] The present invention has found that VEGF-B protein and VEGF-B protein active peptides can reduce the number of ocular inflammatory cells and inhibit the expression of inflammatory factors, have anti-acute anterior uveitis effects, and can inhibit macrophage M1 polarization. They can be used to prepare drugs for preventing and treating acute anterior uveitis or drugs for inhibiting macrophage M1 polarization, provide new ideas for the prevention and treatment of eye diseases, and have very broad application prospects in the medical field. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the mRNA expression level of VEGF-B in the iris-ciliary body complex of mice in the normal group and acute anterior uveitis model group.

[0020] Figure 2 The knockout of VEGF-B gene promotes the inflammatory response of acute anterior uveitis. - / - ), VEGF-B mRNA expression levels in the iris-ciliary complex of wild-type mice (WT) of the same litter; B is the VEGF-B gene knockout mice (Vegf-b - / - ) and wild-type mice (WT) of the same litter under slit lamp microscope; C is the VEGF-B gene knockout mice (Vegf-b - / -), wild-type mice (WT) of the same litter; D and E are the clinical severity scores of VEGF-B gene knockout mice (Vegf-b - / - ), hematoxylin-eosin staining (HE staining) and quantification of eyeball sections of wild-type mice (WT) of the same littermate; F and G are VEGF-B gene knockout mice (Vegf-b - / - ), fluorescent staining results and quantification of eyeball sections of wild-type mice (WT) from the same littermates.

[0021] Figure 3 The results of VEGF-B protein inhibiting macrophage M1 polarization. A is the relative expression level of mRNA of inflammatory factors TNF-α, IL-6, and IL-1β in THP-1 cells with siRNA knockdown of VEGF-B expression (si-VEGF-B) and normal THP-1 cells (si-NC); B is the relative expression level of mRNA of VEGF-B gene knockout mice (Vegf-b - / - ) The mRNA expression levels of inflammatory factors TNF-α, IL-6, and IL-1β in peritoneal macrophages from wild-type mice (WT) and those from the same littermates; C, D, and E are the mRNA expression levels of inflammatory factors IL-1β, IL-6, and TNF-α in THP-1 cells treated with lipopolysaccharide (LPS), bovine serum albumin (BSA), and VEGF-B protein, respectively.

[0022] Figure 4 This is the result of VEGF-B protein specifically inhibiting inflammatory response.

[0023] Figure 5 It is the result of VEGF-B protein and its active peptide segments inhibiting inflammatory response. 167 A is the specific structural region of THP-1 cells stimulated by LPS and B is the mRNA expression levels of inflammatory factors TNF-α, IL-6, and IL-1β after the addition of VEGF-B protein, Peptide1, Peptide2, Peptide3, Peptide4, and Peptide5 respectively.

[0024] Figure 6The results of VEGF-B protein treatment of acute anterior uveitis. A shows the expression of VEGF-B protein in the iris-ciliary complex of AAV-VEGF-B group mice and AAV-GFP group mice treated with LPS; B shows the results of slit lamp microscope observation of AAV-VEGF-B group mice and AAV-GFP group mice treated with LPS; C shows the clinical severity score of AAV-VEGF-B group mice and AAV-GFP group mice treated with LPS; D shows the flow cytometry detection result of AAV-VEGF-B group mice and AAV-GFP group mice treated with LPS; E and F show the hematoxylin-eosin staining (HE staining) and quantification of eyeball sections of AAV-VEGF-B group mice and AAV-GFP group mice treated with LPS; G and H show the results of fluorescence staining and quantification of eyeball sections of AAV-VEGF-B group mice and AAV-GFP group mice treated with LPS. DETAILED DESCRIPTION

[0025] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0026] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods, and the reagents and materials used are all commercially available unless otherwise specified.

[0027] Example 1 VEGF-B expression is correlated with acute anterior uveitis

[0028] 1. Modeling of acute anterior uveitis

[0029] Mice (C57 mice purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.) were injected with lipopolysaccharide (LPS) into the vitreous cavity as the acute anterior uveitis model group (EIU), and mice without intravitreal LPS injection were used as the normal group.

[0030] The EIU model was established as follows: mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (dose 10 mL / kg), tropicamide was used to dilate the pupil, procainamide was used for corneal surface anesthesia, and sodium carboxymethylcellulose was used to prevent corneal drying. LPS (1 μL / eye, 12.5 ng) was injected intravitreally through a 0.2 mm diameter puncture behind the mouse corneal limbus using a sterile 5 μL syringe (Hamilton, 7633-01) and a 33-gauge blunt needle (Hamilton, 7803-05).

[0031] After LPS was injected into the mouse eyes to create the model, the mice showed symptoms such as tearing, eye secretions, pupil constriction, umbilical congestion and edema, indicating that the acute uveitis model was successfully created.

[0032] 2. qRT-PCR detection of VEGF-B mRNA expression level in the iris-ciliary complex of mice in the normal group and model group

[0033] 1) Take the iris-ciliary body complex of the modeled mouse, wash it once with PBS, add 500 μL Trizol to lyse it, and transfer it to a 1.5 mL centrifuge tube;

[0034] 2) Add 100 μL of chloroform, shake vigorously for 20 s, let stand for 3 min, and then centrifuge at 4°C for 10 min, 12,000 rpm;

[0035] 3) Pipette the supernatant into a new centrifuge tube (do not aspirate the middle layer), add isopropanol to the supernatant at a volume ratio of 1:1, mix well, let stand for 10 min, and then centrifuge at 4°C for 10 min;

[0036] 4) Discard the supernatant and add 1 mL 75% ethanol;

[0037] 5) Centrifuge at 4℃ for 5 min, discard the supernatant, add 1 mL 75% ethanol, centrifuge at 4℃ for 5 min, discard the supernatant, invert the centrifuge tube on clean absorbent paper, wait until the white precipitate at the bottom of the tube becomes transparent, add 50 μL ddH2O, and store at -20℃;

[0038] 6) Obtain total RNA, perform reverse transcription using the FastKing RT kit with gDNase (TIANGEN) kit, and synthesize cDNA;

[0039] 7) Using the synthesized cDNA as a template, the mRNA expression level of VEGF-B was detected on an ABI QuantStudio6 Flex device (Life Technologies) PCR instrument according to the SYBR Green (ROCHE) kit;

[0040] 8) The expression of each gene was calculated using the delta-delta Ct method using 18S rRNA as the internal reference gene. The primer sequences of each gene were obtained from the Primerbank website.

[0041] Table 1 qPCR primer sequences for each gene

[0042]

[0043] The results of qRT-PCR were as follows Figure 1 As shown. Figure 1It can be seen that compared with the normal group, the mRNA expression level of VEGF-B in the iris-ciliary body complex of the model group mice was significantly lower, which shows that the expression of VEGF-B is related to acute anterior uveitis.

[0044] Example 2 Knockout of VEGF-B gene can promote the inflammatory response in acute anterior uveitis

[0045] 1. Acute anterior uveitis model: VEGF-B knockout mice (Vegf-b - / - ) and wild-type mice (WT) of the same littermate were injected with LPS (1 μL / eye, 12.5 ng). - / - ) and wild-type mice (WT) of the same litter were constructed by Jackson Laboratory, and the parents' genotypes were het×het and obtained by co-breeding.

[0046] 2. qRT-PCR detection of VEGF-B knockout mice (Vegf-b - / - ) and the mRNA expression level of VEGF-B in the iris-ciliary complex of wild-type mice (WT) of the same littermate. The results of qRT-PCR detection are shown in Figure 2 As shown in A. Figure 2 As shown in Figure A, VEGF-B knockout mice (Vegf-b - / - ) had a significantly lower relative expression level of VEGF-B mRNA in the iris-ciliary body complex.

[0047] 3. Observation of VEGF-B knockout mice (Vegf-b - / - ) and the inflammatory phenotype of the anterior segment of the eyes of wild-type mice (WT)

[0048] VEGF-B gene knockout mice (Vegf-b - / - ) and the anterior segment of the eyes of wild-type mice (WT) in the same litter were observed under a slit lamp microscope. Figure 2 As shown in B (scale bar: 200 μm). Figure 2 As shown in B, compared with wild-type mice (WT) in the same littermate, VEGF-B knockout mice (Vegf- / - ) showed a more severe inflammatory phenotype, including more severe pupil constriction caused by more severe posterior synechiae (the pupil is circled by the white dotted line), more inflammatory cells and pigment cells attached to the posterior cornea (indicated by the yellow arrow), more severe ciliary congestion (indicated by the white arrow), and more severe pus in the anterior chamber of the eyeball (circled by the yellow dotted line).

[0049] 4. According to the clinical scoring criteria for acute anterior uveitis, the VEGF-B gene knockout mice (Vegf-b - / - ) and wild-type mice (WT) from the same litter.

[0050] VEGF-B gene knockout mice (Vegf-b - / - ), clinical severity scores of wild-type mice in the same litter as Figure 2 As shown in C. Figure 2 As shown in Figure C, compared with wild-type mice (WT) in the same littermate, VEGF-B knockout mice (Vegf- - / - ) had higher clinical severity scores and presented a more severe inflammatory phenotype.

[0051] 5. VEGF-B knockout mice (Vegf-b - / - ), and the eyeball sections of wild-type mice (WT) of the same littermate were stained with hematoxylin-eosin (HE) and immunofluorescence staining.

[0052] Hematoxylin-eosin staining (HE staining) was performed as follows: dewaxing in xylene (SCRC, 10023418) for 20 minutes, dewaxing in xylene II (SCRC, 10023418) for 20 minutes, dewaxing in 100% ethanol I (SCRC, 100092683) for 5 minutes, dewaxing in 100% ethanol II (SCRC, 100092683) for 5 minutes, and dewaxing in 75% ethanol for 5 minutes. The eyeball sections were then stained with hematoxylin solution (Servicebio, G1003) for 5 minutes, rinsed with tap water, and then stained with hematoxylin differentiation solution (Servicebio, G1003), and rinsed with tap water. Treated with hematoxylin-scott tap blue (Servicebio, G1003), rinsed with tap water, treated with 85% ethanol for 5 minutes, 95% ethanol for 5 minutes, and then treated with eosin dye (Servicebio, G1003) for 5 minutes. After dehydration, sections were sealed with neutral glue (SCRC, 10004160). Images were captured using an AX10 Imager Z2 (Zeiss) microscope.

[0053] Immunofluorescence staining was performed as follows: eyeball sections were incubated in 0.5% Triton X-100 (Sigma, X100) prepared in 1× PBS for 15 minutes, then blocked with 5% normal goat serum for 1 hour, and incubated with primary antibodies overnight at 4°C. Sections were washed three times with 1× PBS, incubated with secondary antibodies for 1 hour at room temperature, and then incubated with DAPI for 5 minutes for nuclear staining (Sigma, D9542). Images were taken with an AX10 Imager Z2 (Zeiss) microscope and analyzed using ZEN 2012 (Zeiss) and ImageJ.

[0054] VEGF-B knockout mice (Vegf-b - / - ), and the HE staining results of the eyeball sections of wild-type mice (WT) of the same litter are as follows Figure 2 As shown in D (scale bar: 50 μm) and E. Figure 2 As shown in Figures D and E, compared with wild-type mice (WT) in the same littermate, VEGF-B knockout mice (Vegf-b - / - ) showed more CD45-positive cells infiltrating into the anterior chamber (indicated by the yellow arrow) and more fibrin exuding (indicated by **).

[0055] VEGF-B knockout mice (Vegf-b - / - ), and the results of immunofluorescence staining of eyeball sections of wild-type mice (WT) of the same litter are shown in Figure 2 As shown in F (scale bar: 50 μm) and G. Figure 2 As shown in Figures F and G, compared with wild-type mice (WT) in the same littermate, VEGF-B knockout mice (Vegf-b - / - ) had significantly more CD45-positive cells.

[0056] This suggests that VEGF-B dysfunction promotes the inflammatory response in acute anterior uveitis.

[0057] Example 3 VEGF-B protein has the effect of inhibiting macrophage M1 polarization

[0058] 1. siRNA knocked down the expression of VEGF-B in THP-1 cells (purchased from Zhongqiao Xinzhou Biotechnology Co., Ltd., catalog number: ZQ0086). Then qRT-PCR was used to detect the expression levels of inflammatory factors TNF-α, IL-6, and IL-1β in THP-1 cells with siRNA knocked down VEGF-B expression (si-VEGF-B) and normal THP-1 cells (si-NC). The qRT-PCR test results are as follows Figure 3 As shown in A. Figure 3 As shown in A, compared with normal THP-1 cells (si-NC), the expression levels of inflammatory factors TNF-α, IL-6, and IL-1β in THP-1 cells (si-VEGF-B) with siRNA knockdown of VEGF-B expression were significantly higher. This indicates that knocking down VEGF-B protein expression can significantly promote the expression of inflammatory factors in cells, that is, promote the polarization of macrophages to the M1 subtype.

[0059] The method for knocking down VEGF-B expression in THP-1 cells by siRNA was as follows:

[0060] (1) 1×10 THP-1 cells were seeded in each well of a 6-well plate.6 After inducing adhesion, configure the transfection system according to the following scheme:

[0061] Solution A: Opti-MEM 100 µL, add RNAiMax 6 µL, mix gently, and let stand at room temperature for 5 min;

[0062] Solution B: Opti-MEM 100 μL, add 3 μL of siRNA with a concentration of 20 μM (the sequence of siRNA is as follows: GCTTAGAGCTCAACCCAGA), mix gently, and let stand at room temperature for 5 min;

[0063] (2) Gently mix Solution A and Solution B and let stand at room temperature for 10 min to obtain the transfection solution;

[0064] (3) Dilute the transfection solution in step (2) to 1.5 mL with Opti-MEM and add it to the above 6-well plate;

[0065] (4) Place the 6-well plate in an incubator for 6 h and replace with full culture medium. Perform subsequent experiments 48 h later.

[0066] 2. LPS treatment of VEGF-B knockout mice (Vegf-b - / - ) peritoneal macrophages and peritoneal macrophages of wild-type mice (WT) of the same littermate. Then qRT-PCR was used to detect the expression of VEGF-B knockout mice (Vegf-b / - The mRNA expression levels of inflammatory factors TNF-α, IL-6, and IL-1β in peritoneal macrophages of wild-type mice (WT) and peritoneal macrophages of the same littermate. Figure 3 As shown in B. Figure 3 As shown in B, compared with the peritoneal macrophages of wild-type mice (WT) treated with LPS, the macrophages of VEGF-B knockout mice (Vegf-b - / - ) The mRNA expression levels of inflammatory factors TNF-α, IL-6, and IL-1β in peritoneal macrophages were significantly higher.

[0067] The extraction and culture methods of mouse peritoneal macrophages (PCMs) are as follows:

[0068] (1) Preparation of FTG solution

[0069] 4 g FTG (Fluid Thioglycollate Medium; Manufacturer: Sigma; Product Number: T9032; Specification: 1 kg) + 100 mL H2O → sterilize with high pressure steam → refrigerate at 4°C for later use.

[0070] (2) Intraperitoneal injection of 6- to 8-week-old mice (2 mL FTG solution / mouse)

[0071] Grasp the mouse with your left hand, with its abdomen facing upwards, and with your right hand, insert the injection needle into the subcutaneous tissue at the left lower abdomen. Push the needle forward 0.5 cm, then pass it through the abdominal muscle at a 45-degree angle. Fix the needle and slowly inject the drug solution. To avoid damaging the internal organs, keep the mouse in a head-down position and move the internal organs to the upper abdomen.

[0072] (3) Two days later, the mice were killed and peritoneal macrophages were obtained.

[0073] 1) Mice were killed by cervical dislocation;

[0074] 2) Soak or scrub the abdomen with 75% alcohol and place it on the workbench with the ventral surface facing up;

[0075] 3) Use small forceps to lift the mouse's lower abdominal skin, make a small cut, and tear the skin up and down to fully expose the peritoneum;

[0076] 4) Use a 10 mL syringe to inject 8 mL of 1640 medium → Shake and massage the mouse abdomen appropriately;

[0077] 5) Change to a smaller needle with the needle tip pointing upwards, avoiding the intestine and fat, and draw out about 6 mL of peritoneal fluid;

[0078] 6) Repeat peritoneal lavage (steps 4-5);

[0079] 7) Centrifuge the cell suspension and discard the supernatant;

[0080] 8) Resuspend in appropriate amount of 1640 complete culture medium and count cells;

[0081] 9) After counting the number, plate the cells in a 6-well plate at 2×10 6 Pieces / per hole;

[0082] 10) After culturing at 37°C for 2 hours, wash once with 1640 culture medium and then replace the medium to remove cells that are not attached to the wall.

[0083] 3. Treat THP-1 cells with LPS and add VEGF-B recombinant protein to the cells. Then qRT-PCR was used to detect the mRNA expression levels of inflammatory factors TNF-α, IL-6, and IL-1β in THP-1 cells after LPS treatment. The results of qRT-PCR are shown in the figure. Figure 3 As shown in CE. Figure 3CE in the Figure shows that when control bovine serum albumin (BSA) was added to THP-1 cells treated with LPS, the expression levels of inflammatory factors TNF-α, IL-6, and IL-1β in the cells were higher; while when VEGF-B recombinant protein was added to THP-1 cells treated with LPS, the expression levels of inflammatory factors TNF-α, IL-6, and IL-1β in the cells were lower. This indicates that VEGF-B protein can effectively inhibit the polarization of pro-inflammatory M1 subtype of macrophages.

[0084] Example 4 VEGF-B protein has specificity in inhibiting inflammatory response

[0085] 1. Extract bone marrow-derived macrophages (BMDMs) from wild-type mice: After anesthesia, mice were killed by dislocation. The mice were placed in a beaker containing sufficient 75% ethanol and soaked for disinfection for 5 minutes. The tibia and femur of the mice were separated on a sterile operating table. The bone marrow was flushed out of the tibia and femur with pre-cooled culture medium, and the washing was repeated 3 times until no obvious red color was seen in the leg bones. The culture medium containing bone marrow cells was repeatedly blown with a 5 mL pipette to disperse the cell clumps, and then the cells were sieved with a 70μm cell filter and transferred to a 15 mL centrifuge tube. Centrifuged at 1500 rpm / min for 5 minutes, the supernatant was discarded, and the red blood cell lysis buffer was added to resuspend and stand for 5 minutes. After centrifugation at 1500 rpm / min for 5 minutes, the supernatant was discarded and resuspended with cold prepared bone marrow macrophage induction culture medium, and plated (2×10 6 On the third day of culture, half of the bone marrow macrophage induction medium was replaced, and on the fifth day, all of the medium was replaced. On the seventh day, the medium was used for subsequent experiments.

[0086] 2. LPS stimulated mouse bone marrow macrophages for 6 h, and then VEGF-B recombinant protein (100-20B, Peprotech) and VEGF-A recombinant protein (100-20, Peprotech) were added to the cells and incubated for 6 hours. qRT-PCR was used to detect the mRNA expression levels of inflammatory factors TNF-α, IL-6, and IL-1β in mouse bone marrow macrophages. The results of qRT-PCR are shown in Figure 2. Figure 4 As shown. Figure 4 It can be seen that VEGF-B protein showed a significant function of inhibiting inflammatory response, while VEGF-A protein had no obvious effect of inhibiting inflammatory response. This shows that VEGF-B protein has specificity in inhibiting inflammatory response.

[0087] Example 5 Specific structural region of VEGF-B protein that inhibits inflammation

[0088] 1. VEGF-B 167The specific structural regions of protein active peptides Peptide1, Peptide2, Peptide3, Peptide4, and Peptide5 are as follows Figure 5 As shown in A, the sequence is shown in Table 2. Figure 5 From A in the figure, we can see that Peptide 1, Peptide 2, and Peptide 3 are located in the VEGFR1 binding region (KDR binding region), and Peptide 4 and Peptide 5 are located in the NRP1 binding region.

[0089] Table 2 VEGF-B 167 Protein active peptide sequence list

[0090]

[0091] 2. Normal THP-1 cells were used as the control group (CTL group), THP-1 cells stimulated with LPS were used as the LPS group, VEGF-B full-length protein was added to the cells stimulated with LPS for 6 h as the VB FL group, VEGF-B protein active peptide Peptide1 was added to the cells stimulated with LPS for 6 h as the Peptide1 group, Peptide2 was added to the cells stimulated with LPS for 6 h as the Peptide2 group, Peptide3 was added to the cells stimulated with LPS for 6 h as the Peptide3 group, Peptide4 was added to the cells stimulated with LPS for 6 h as the Peptide4 group, and Peptide5 was added to the cells stimulated with LPS for 6 h as the Peptide5 group.

[0092] qRT-PCR was used to detect the mRNA expression levels of inflammatory factors TNF-α, IL-6, and IL-1β in the CTL group, LPS group, VB FL group, Peptide 1 group, Peptide 2 group, Peptide 3 group, Peptide 4 group, and Peptide 5 group. Figure 5 As shown in B, Figure 5As shown in B, compared with the CTL group, the expression levels of inflammatory factors TNF-α, IL-6, and IL-1β in the LPS group were significantly increased. Compared with the LPS group, the expression levels of inflammatory factors TNF-α, IL-6, and IL-1β in cells after adding VEGF-B full-length protein, Peptide1, Peptide2, Peptide3, Peptide4, and Peptide5 decreased. This shows that VEGF-B full-length protein, Peptide1, Peptide2, Peptide3, Peptide4, and Peptide5 all showed a certain degree of anti-inflammatory effect, among which Peptide5 located in the NRP1 binding region showed the most significant anti-inflammatory effect.

[0093] Example 6 VEGF-B protein has the effect of treating acute anterior uveitis

[0094] 1. Inject VEGF-B into the vitreous cavity of mice 167 The adeno-associated virus expressing VEGF protein (purchased from Shandong Weizhen Biotechnology Co., Ltd.) was used as the AAV-VEGF-B group, and the adeno-associated virus expressing GFP was injected into the vitreous cavity of mice as the AAV-GFP group.

[0095] 2. Western blotting to detect the expression of VEGF-B protein in the iris-ciliary complex of mice in the AAV-VEGF-B group and the AAV-GFP group

[0096] Western blotting results Figure 6 As shown in A. Figure 6 As shown in A, compared with the AAV-GFP group, the VEGF-B protein was overexpressed in the iris-ciliary body complex of the AAV-VEGF-B group mice.

[0097] 3. Acute anterior uveitis model: AAV-VEGF-B group mice and AAV-GFP group mice were treated with LPS.

[0098] 4. Observation of the inflammatory phenotype of the anterior segment of the eyes of the AAV-VEGF-B group and the AAV-GFP group mice after modeling under a slit lamp microscope

[0099] The results of observation of the anterior segment of the eyes of the AAV-VEGF-B group mice and the AAV-GFP group mice under a slit lamp microscope after modeling are as follows Figure 6 As shown in B (scale bar: 200 μm). Figure 6As shown in B, the mice in the AAV-GFP group after modeling showed more severe inflammatory phenotypes, including more severe pupil constriction caused by more severe posterior synechiae (the pupil is circled by the white dotted line), more severe ciliary congestion (indicated by the white arrow) and more severe pus in the anterior chamber of the eye (circled by the yellow dotted line). The inflammatory phenotypes of the mice in the AAV-VEGF-B group after modeling were significantly improved, including normal pupils (indicated by the white dotted line), transparent and clean corneas, and no obvious ciliary congestion (indicated by the white arrow) and pus in the anterior chamber of the eye (indicated by the yellow dotted line).

[0100] 5. Scoring of the AAV-VEGF-B group mice and the AAV-GFP group mice after modeling according to the clinical scoring criteria for acute anterior uveitis

[0101] Clinical severity scores Figure 6 As shown in C. Figure 6 As shown in C, compared with the AAV-GFP group mice, the AAV-VEGF-B group mice had lower clinical severity scores and showed a milder inflammatory phenotype.

[0102] 6. Flow cytometry detection of the eyes of the AAV-VEGF-B group and AAV-GFP group mice after modeling

[0103] (1) Preparation of single-cell suspension of mouse anterior segment tissue: Immerse the mouse eyeball in RPIM-1640 containing 10% FBS, and use eye scissors to remove the periocular muscles and fascia under a microscope. Make a circular incision along the edge of the cornea and sclera to separate the anterior and posterior chamber tissues. Use a 200 μL pipette tip to gently aspirate the infiltrating cells in the vitreous cavity, and remove the lens, retina, and choroid. Use forceps to separate the iris from the ciliary body-cornea complex, gently tear it into small pieces, and transfer the culture medium into a 15 mL centrifuge tube. Centrifuge at 50 g for 1 min, transfer the culture supernatant to another clean 15 mL centrifuge tube, wash the remaining tissue twice with PBS, then add 500 μL 7 mg / mL type I collagenase, place at 37°C for 20 min, add 3 mg / mL DNase I enzyme, continue digestion at 37°C for 10 min, and add the culture medium collected in the previous step to inactivate the enzyme activity. Use a 1 mL pipette tip to gently blow the tissue through a 70 μm and 40 μm cell sieve in turn.

[0104] (2) Flow cytometry: The single cell suspension prepared above was washed twice with PBS containing 1% BSA, incubated with CD16 / 32 antibody on ice for 20 min (1:200), and the target antibody was added and incubated on ice for 30 min. Protected from light, the cells were washed three times with PBS containing 1% BSA, and stained with PI. The cells were washed three times with PBS containing 1% BSA, resuspended and tested, and the data were analyzed with flowJo software.

[0105] Flow cytometry results of single cell suspensions of mouse anterior chamber tissues (cornea, iris, ciliary body, and infiltrating cells) are shown in Figure 2. Figure 6 As shown in D. Figure 6 As shown in D, the proportion of inflammatory cells in the anterior chamber of the AAV-VEGF-B group mice was significantly reduced compared with that of the AAV-GFP group mice.

[0106] 7. HE staining and immunofluorescence staining were performed on the eyeball sections of the AAV-VEGF-B group mice and the AAV-GFP group mice after modeling.

[0107] HE staining results of eyeball sections of mice in the AAV-VEGF-B group and the AAV-GFP group are shown in Figure 6 As shown in E (scale bar: 50 μm) and F. Figure 6 As shown in E and F, the number of CD45-positive cells infiltrating in the anterior chamber of mice in the AAV-VEGF-B group was significantly reduced, and the fibrin exudation in the anterior chamber (shown by **) was significantly reduced.

[0108] The results of immunofluorescence staining of eyeball sections of mice in the AAV-VEGF-B group and the AAV-GFP group are shown in Figure 6 As shown in G (scale bar: 50 μm) and H. Figure 6 As shown in G and H, compared with the AAV-GFP group, the number of CD45-positive cells infiltrating in the anterior chamber of the AAV-VEGF-B group mice was significantly less. This indicates that VEGF-B protein can effectively inhibit the infiltration of inflammatory cells in the eye and protect the integrity of the blood-aqueous barrier, that is, VEGF-B protein has the effect of treating acute anterior uveitis.

[0109] The above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention is described in detail with reference to the above embodiments, ordinary technicians in the relevant field should understand that the technical solutions of the present invention can be modified or equivalently replaced, but these modifications or replacements are all within the protection scope of the present invention.

Claims

1. VEGF-B 167 Application of protein in preparing medicine for treating acute anterior uveitis.

2. The use according to claim 1, characterized in that The drug for treating acute anterior uveitis is a drug for reducing ocular inflammatory cells.

3. The use according to claim 2, characterized in that The inflammatory cells include CD45 positive cells.

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