WO2025168167A1 - Use of vegf-b protein and vegf-b protein active peptide fragment in preparation of drug for preventing and/or treating acute anterior uveitis - Google Patents

Abstract

The use of a VEGF-B protein and a VEGF-B protein active peptide fragment in the preparation of a drug for preventing and/or treating acute anterior uveitis. The VEGF-B protein and the VEGF-B protein active peptide fragment have a good drug safety and an anti-acute anterior uveitis effect, can reduce the number of ocular inflammatory cells and inhibit the expression of inflammatory factors, and can inhibit macrophage M1 polarization.

Description

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

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 technology:

Vascular endothelial growth factor (VEGF-B) is a member of the VEGF family. It primarily 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 respond to VEGF-B by increasing the expression and activity of plasminogen activator inhibitor 1 (PAI-1) and urokinase-type plasminogen activator (uPA). NRP-1, a receptor for cerebral adenomatous proteins/signalins, guides neuronal growth cones and selectively binds to VEGF-B to exert its effects.

Uveitis is an inflammatory disease of the eye, primarily affecting the iris, ciliary body, and choroid. Severe cases can even lead to blindness, accounting for 10% to 15% of blindness worldwide and approximately 4% to 10% of blindness in China. Acute anterior uveitis (AAU) is the most common type of uveitis, with an incidence of approximately 28% to 61%. Clinical manifestations of AAU include ciliary congestion, pronounced anterior chamber flare, sudden eye pain, redness, photophobia, and blurred vision. Severe cases can develop or worsen posterior synechiae and glaucoma, leading to irreversible vision loss or damage. Clinically used drugs for the treatment of anterior uveitis include: 1) mydriatics and ciliary muscle paralytics, which can only relieve clinical symptoms such as pain and photophobia; 2) non-steroidal anti-inflammatory drugs, which are only effective for certain non-necrotizing anterior scleral inflammations, and systemic use increases the risk of gastrointestinal ulcers; 3) steroids, the main drugs for the treatment of uveitis, are divided into topical and systemic administration and have serious side effects. Ocular side effects include cataracts, increased intraocular pressure, and glaucoma, and systemic side effects include diabetes, hypertension, peptic ulcers, insomnia, and osteoporosis; 4) immunomodulatory therapies, such as anti-TNF-α, anti-IL-6, and anti-IL-1β therapies, can achieve better results when combined with traditional steroids or cellular immunosuppressants, but infections caused by immunosuppression are the main side effect. Because current drugs used in the treatment of acute anterior uveitis can only control inflammation to a certain extent and have serious side effects that can further lead to vision loss, there is an urgent need for safe and effective targeted drugs to prevent and treat acute anterior uveitis.

Summary of the invention:

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.

According to one aspect of the present invention, the use of a VEGF-B protein or an active peptide fragment of the VEGF-B protein in the preparation of a medicament for preventing and/or treating acute anterior uveitis is provided. The VEGF-B protein and active peptide fragment of the VEGF-B protein provided by the present invention have excellent anti-inflammatory effects, can reduce the number of inflammatory cells in the eye and inhibit the expression of inflammatory factors, thereby playing a role in treating acute anterior uveitis, providing new ideas for the prevention and treatment of eye diseases, and has very broad application prospects in the medical field.

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

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

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.

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

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.

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

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

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.

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

Preferably, the VEGF-B protein comprises VEGF-B167.

According to another aspect of the present invention, a medicine is provided, which comprises VEGF-B protein and/or VEGF-B protein active peptide segment as an active ingredient.

The present invention has the following beneficial effects:

The present invention has found that VEGF-B protein and VEGF-B protein active peptide fragments can reduce the number of inflammatory cells in the eye 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, providing new ideas for the prevention and treatment of eye diseases and having very broad application prospects in the medical field.

BRIEF DESCRIPTION OF THE DRAWINGS

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

Figure 2 shows the results of VEGF-B gene knockout promoting the inflammatory response in acute anterior uveitis. A shows the mRNA expression level of VEGF-B in the iris-ciliary complex of VEGF-B knockout mice (Vegf-b ^-/- ) and their wild-type littermates (WT); B shows the slit lamp microscopic observation of VEGF-B knockout mice (Vegf-b ^-/- ) and their wild-type littermates (WT) after modeling; C shows the clinical severity score of VEGF-B knockout mice (Vegf-b ^-/- ) and their wild-type littermates (WT); D and E show the hematoxylin-eosin (HE) staining and quantification of eye sections of VEGF-B knockout mice (Vegf-b ^-/- ) and their wild-type littermates (WT); F and G show the fluorescence staining and quantification of eye sections of VEGF-B knockout mice (Vegf-b ^-/- ) and their wild-type littermates (WT) after modeling.

Figure 3 shows the results of VEGF-B protein inhibiting macrophage M1 polarization. A shows the relative mRNA expression levels 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 shows the mRNA expression levels of inflammatory factors TNF-α, IL-6, and IL-1β in peritoneal macrophages from VEGF-B knockout mice (Vegf-b ^-/- ) and wild-type littermates (WT) after LPS treatment; C, D, and E show 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.

FIG4 shows the results of VEGF-B protein specifically inhibiting inflammatory response.

Figure 5 shows the results of VEGF-B protein and its active peptides inhibiting inflammatory responses. A shows the specific structural region of VEGF-B167; B shows the mRNA expression levels of inflammatory factors TNF-α, IL-6, and IL-1β in LPS-stimulated THP-1 cells after the addition of VEGF-B protein, Peptide 1, Peptide 2, Peptide 3, Peptide 4, and Peptide 5, respectively.

Figure 6 shows the results of VEGF-B protein treatment for acute anterior uveitis. A shows the expression of VEGF-B protein in the iris-ciliary complex of LPS-treated AAV-VEGF-B and AAV-GFP mice; B shows the results of slit lamp microscopy observation of LPS-treated AAV-VEGF-B and AAV-GFP mice; C shows the clinical severity scores of LPS-treated AAV-VEGF-B and AAV-GFP mice; D shows the flow cytometry results of LPS-treated AAV-VEGF-B and AAV-GFP mice; E and F show the hematoxylin-eosin (HE) staining and quantification of eye sections of LPS-treated AAV-VEGF-B and AAV-GFP mice; G and H show the fluorescence staining and quantification of eye sections of LPS-treated AAV-VEGF-B and AAV-GFP mice.

Specific implementation method:

The following examples are provided to further illustrate the present invention, but are not intended to limit the present invention.

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

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

1. Acute anterior uveitis modeling

Mice (C57 mice purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were injected with lipopolysaccharide (LPS) into the vitreous cavity to form the acute anterior uveitis model group (EIU), and mice without LPS injection into the vitreous cavity served as the normal group.

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

After LPS injection into the mouse eye, the mouse showed tearing, eye discharge, and pupil constriction. Symptoms such as congestion and edema were observed, indicating that the acute uveitis model was successfully established.

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

1) After modeling, the iris-ciliary body complex of the mouse was removed, washed once with PBS, lysed with 500 μL Trizol, and transferred to a 1.5 mL centrifuge tube.

2) Add 100 μL of chloroform, shake vigorously for 20 seconds, let stand for 3 minutes, and then centrifuge at 12,000 rpm for 10 minutes at 4°C;

3) Pipette the supernatant into a new centrifuge tube (avoiding the middle layer), add isopropanol to the supernatant at a 1:1 volume ratio, mix well, let stand for 10 minutes, and then centrifuge at 4°C for 10 minutes;

4) Discard the supernatant and add 1 mL of 75% ethanol;

5) Centrifuge at 4°C for 5 minutes, discard the supernatant, add 1 mL of 75% ethanol, centrifuge at 4°C for 5 minutes, discard the supernatant, and invert the centrifuge tube on clean absorbent paper. When the white precipitate at the bottom of the tube becomes transparent, add 50 μL of dd H ₂ O and store at -20°C.

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

7) Using the synthesized cDNA as a template, the VEGF-B mRNA expression level was detected on an ABI QuantStudio 6Flex device (Life Technologies) using the SYBR Green (ROCHE) kit.

8) Gene expression was calculated using the delta-delta Ct method, using 18S rRNA as the internal reference gene. Primer sequences for each gene were obtained from PrimerBank.

Table 1 qPCR primer sequences for each gene

The qRT-PCR test results are shown in Figure 1. As shown in Figure 1, the expression level of VEGF-B mRNA in the iris-ciliary complex of mice in the model group was significantly lower than that in the normal group. This suggests that VEGF-B expression is associated with acute anterior uveitis.

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

1. Acute Anterior Uveitis Model: LPS (12.5 ng/eye) was injected intravitreally into VEGF-B knockout mice (Vegf-b ^-/- ) and their wild-type littermates (WT). VEGF-B knockout mice (Vegf-b ^-/- ) and their wild-type littermates (WT) were generated by Jackson Laboratory and co-bred from het × het parents.

2. qRT-PCR was used to detect VEGF-B mRNA expression levels in the iris-ciliary complex of VEGF-B knockout mice (Vegf-b ^-/- ) and their wild-type littermates (WT). The qRT-PCR results are shown in Figure 2, A. As shown in Figure 2, A, the relative expression level of VEGF-B mRNA in the iris-ciliary complex of VEGF-B knockout mice (Vegf-b ^-/- ) was significantly lower than that of their wild-type littermates (WT).

3. Observation of the inflammatory phenotype of the anterior segment of the VEGF-B gene knockout mice (Vegf-b ^-/- ) and the wild-type mice (WT) in the same littermate under a slit lamp microscope

Slit lamp microscopic observation of the anterior segment of the VEGF-B knockout mice (Vegf-b ^-/- ) and their wild-type littermates (WT) after modeling is shown in Figure 2B (scale bar: 200 μm). As shown in Figure 2B, the VEGF-B knockout mice (Vegf-b ^/- ) exhibited a more severe inflammatory phenotype compared with their wild-type littermates (WT), including more severe pupil constriction caused by 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 eye (circled by the yellow dotted line).

4. Score the VEGF-B knockout mice (Vegf-b ^-/- ) and wild-type mice (WT) after modeling according to the clinical scoring criteria for acute anterior uveitis

The clinical severity scores of VEGF-B knockout mice (Vegf-b ^-/- ) and their wild-type littermates after modeling are shown in Figure 2, C. As shown in Figure 2, C, the clinical severity scores of VEGF-B knockout mice (Vegf-b ^-/- ) were higher than those of their wild-type littermates (WT), indicating a more severe inflammatory phenotype.

5. The eyeball sections of VEGF-B gene knockout mice (Vegf-b ^-/- ) and wild-type mice (WT) were stained with hematoxylin-eosin (HE) and immunofluorescence.

Hematoxylin-eosin staining (HE staining) was performed as follows: dewaxing with xylene (SCRC, 10023418) for 20 minutes, dewaxing with xylene II (SCRC, 10023418) for 20 minutes, dewaxing with 100% ethanol I (SCRC, 100092683) for 5 minutes, dewaxing with 100% ethanol II (SCRC, 100092683) for 5 minutes, and dewaxing with 75% ethanol for 5 minutes. 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. Sections were then treated with hematoxylin-Scott Tap Blue (Servicebio, G1003) and rinsed with tap water. Sections were then treated with 85% ethanol for 5 minutes, 95% ethanol for 5 minutes, and then treated with eosin stain (Servicebio, G1003) for 5 minutes. After dehydration, sections were sealed with neutral gum (SCRC, 10004160). Images were captured using an AX10 Imager Z2 (Zeiss) microscope.

Immunofluorescence staining was performed as follows: eyeball sections were incubated in 0.5% Triton X-100 (Sigma, X100) 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 and incubated with secondary antibodies for 1 hour at room temperature, followed by DAPI (Sigma, D9542) for 5 minutes to stain nuclei. Images were taken with an AX10 Imager Z2 (Zeiss) microscope and analyzed using ZEN 2012 (Zeiss) and ImageJ.

The results of HE staining of eye sections from VEGF-B knockout mice (Vegf-b ^-/- ) and their wild-type littermates (WT) are shown in Figure 2, D (Scale bar: 50 μm) and E. As shown in Figure 2, D and E, the anterior chamber of the VEGF-B knockout mice (Vegf-b ^-/- ) showed more CD45-positive cells infiltrating (indicated by yellow arrows) and more fibrin exuding (indicated by **) than that of their wild-type littermates (WT).

The results of immunofluorescence staining of eye sections from VEGF-B knockout mice (Vegf-b ^-/- ) and their wild-type littermates (WT) are shown in Figure 2, F (Scale bar: 50 μm) and G. As shown in Figure 2, F and G, the number of CD45-positive cells in VEGF-B knockout mice (Vegf-b ^-/- ) was significantly higher than that in their wild-type littermates (WT).

This suggests that VEGF-B functional deficiency promotes the inflammatory response in acute anterior uveitis.

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

1. siRNA knocked down the expression of VEGF-B in THP-1 cells (purchased from Zhongqiao Xinzhou Biotechnology Co., Ltd., catalog number: ZQ0086). qRT-PCR was then used to detect the expression levels of inflammatory factors TNF-α, IL-6, and IL-1β in THP-1 cells (si-VEGF-B) in which siRNA knocked down the expression of VEGF-B and normal THP-1 cells (si-NC). The results of qRT-PCR detection are shown in A in Figure 3. As can be seen from A in Figure 3, the expression levels of inflammatory factors TNF-α, IL-6, and IL-1β in THP-1 cells (si-VEGF-B) in which siRNA knocked down the expression of VEGF-B were significantly higher than those in normal THP-1 cells (si-NC). This shows that knocking down the expression of VEGF-B protein can significantly promote the expression of inflammatory factors in cells, that is, promote the polarization of macrophages to the M1 subtype.

The method for knocking down VEGF-B expression in THP-1 cells by siRNA is as follows:

(1) 1×10 ⁶ THP-1 cells were seeded in each well of a 6-well plate. After induction of adherence, the transfection system was prepared according to the following scheme:

Solution A: Add 100 μL of Opti-MEM to 6 μL of RNAiMax, mix gently, and let stand at room temperature for 5 minutes;

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

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

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

(4) Incubate the 6-well plate in an incubator for 6 hours, then replace the culture medium with full medium. Perform subsequent experiments 48 hours later.

2. LPS was used to treat peritoneal macrophages from VEGF-B knockout mice (Vegf-b ^-/- ) and peritoneal macrophages from wild-type mice (WT) of the same littermates. qRT-PCR was then used to detect the mRNA expression levels of inflammatory factors TNF-α, IL-6, and IL-1β in peritoneal macrophages from VEGF-B knockout mice (Vegf-b ^/- ) and peritoneal macrophages from wild-type mice (WT) of the same littermates after LPS stimulation. The results of qRT-PCR detection are shown in Figure 3B. As can be seen from Figure 3B, the mRNA expression levels of inflammatory factors TNF-α, IL-6, and IL-1β in peritoneal macrophages from VEGF-B knockout mice (Vegf-b ^-/- ) after LPS treatment were significantly higher than those in peritoneal macrophages from wild-type mice (WT) of the same littermates after LPS treatment.

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

(1) Preparation of FTG solution

4 g FTG (Fluid Thioglycollate Medium; Manufacturer: Sigma; Product No.: T9032; Specification: 1 kg) + 100 mL H ₂ O → Autoclave → Refrigerate at 4°C until use.

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

Grab the mouse with your left hand, with its abdomen facing upwards. Use your right hand to insert the injection needle into the subcutaneous tissue at the lower left 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.

(3) Two days later, the mice were killed and peritoneal macrophages were collected.

1) Mice were killed by cervical dislocation;

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

3) Use small forceps to lift the mouse's lower abdominal skin, make a small incision, and tear the skin apart to fully expose the peritoneum.

4) Use a 10 mL syringe to inject 8 mL of 1640 medium. Shake and massage the mouse's abdomen.

5) Change to a smaller needle, with the needle tip pointing upward, avoiding the intestine and fat, and draw out approximately 6 mL of peritoneal fluid;

6) Repeat peritoneal lavage (steps 4-5) once;

7) Centrifuge the cell suspension and discard the supernatant;

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

9) After counting the cells, plate them in a 6-well plate at a density of 2 × 10 ⁶ cells per well.

10) After culturing at 37°C for 2 hours, wash once with 1640 culture medium and then replace the medium to remove non-adherent cells.

3. THP-1 cells were treated with LPS and VEGF-B recombinant protein was added to the cells. qRT-PCR was then 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 qRT-PCR test results are shown in Figure 3C-E. As shown in Figure 3C-E, 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 shows that VEGF-B protein can effectively inhibit the polarization of pro-inflammatory M1 subtype of macrophages.

Example 4 VEGF-B protein is specific for inhibiting inflammatory responses

1. Isolation of bone marrow-derived macrophages (BMDMs) from wild-type mice: Mice were anesthetized and sacrificed by dislocation. The mice were placed in a beaker filled with 75% ethanol and sterilized for 5 minutes. The tibia and femur of the mice were isolated on a sterile operating table. The bone marrow was flushed from the tibia and femur with pre-chilled culture medium, and the washing was repeated three times until no obvious red color was visible in the leg bones. The culture medium containing the bone marrow cells was repeatedly pipetted with a 5 mL pipette to disperse cell clumps. The cells were then filtered through a 70 μm cell strainer and transferred to a 15 mL centrifuge tube. The cells were centrifuged at 1500 rpm/min for 5 minutes, the supernatant discarded, and the tube was resuspended in red blood cell lysis buffer and allowed to stand for 5 minutes. The tube was then centrifuged at 1500 rpm/min for 5 minutes. The supernatant discarded and the tube was resuspended in cold bone marrow macrophage induction medium. The tube was plated (2 × 10 ⁶ viable cells/dish). On the third day of culture, half of the bone marrow macrophage induction medium was replaced, and on the fifth day, the entire medium was replaced. The tube was ready for subsequent experiments on the seventh day.

2. LPS stimulated mouse bone marrow-derived macrophages for 6 hours, 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-derived macrophages. The results of qRT-PCR are shown in Figure 4. As shown in Figure 4, 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 for inhibiting inflammatory response.

Example 5 Specific structural regions of VEGF-B protein that inhibit inflammation

1. The specific structural regions of the active peptides of VEGF-B167 protein, Peptide 1, Peptide 2, Peptide 3, Peptide 4, and Peptide 5, are shown in Figure 5A, and their sequences are shown in Table 2. As shown in Figure 5A, Peptide 1, Peptide 2, and Peptide 3 are located in the VEGFR1 binding region (KDR binding region), while Peptide 4 and Peptide 5 are located in the NRP1 binding region.

Table 2 Sequence list of active peptides of VEGF-B167 protein

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, full-length VEGF-B protein was added to the cells stimulated with LPS for 6 hours as the VB FL group, VEGF-B protein active peptide Peptide1 was added to the cells stimulated with LPS for 6 hours as the Peptide1 group, Peptide2 was added to the cells stimulated with LPS for 6 hours as the Peptide2 group, Peptide3 was added to the cells stimulated with LPS for 6 hours as the Peptide3 group, Peptide4 was added to the cells stimulated with LPS for 6 hours as the Peptide4 group, and Peptide5 was added to the cells stimulated with LPS for 6 hours as the Peptide5 group.

qRT-PCR was used to detect the mRNA expression levels of inflammatory factors TNF-α, IL-6, and IL-1β in the CTL, LPS, VB FL, Peptide 1, Peptide 2, Peptide 3, Peptide 4, and Peptide 5 groups, respectively. The qRT-PCR results are shown in Figure 5B. As shown in Figure 5B, 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 treated with full-length VEGF-B protein, Peptide 1, Peptide 2, Peptide 3, Peptide 4, and Peptide 5 decreased. This shows that the full-length VEGF-B 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.

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

1. Adeno-associated virus expressing VEGF-B167 protein (purchased from Shandong Weizhen Biotechnology Co., Ltd.) was injected into the vitreous cavity of mice as the AAV-VEGF-B group, and adeno-associated virus expressing GFP was injected into the vitreous cavity of mice as the AAV-GFP group.

2. Western blotting was used 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.

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

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

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

The results of slit lamp microscopic observation of the anterior segment of the eyes of the AAV-VEGF-B group and the AAV-GFP group after modeling are shown in Figure 6B (scale bar: 200 μm). As shown in Figure 6B, the AAV-GFP group mice showed more severe inflammatory phenotypes after modeling, including more severe pupil constriction caused by 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 phenotype of the AAV-VEGF-B group mice after modeling was significantly improved, including normal pupils (indicated by the white dotted line), clear and clean corneas, and no obvious ciliary congestion (indicated by the white arrow) and hypopyon (indicated by the yellow dotted line).

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

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

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

(1) Preparation of single-cell suspension of mouse anterior segment tissue: The mouse eyeball was immersed in RPIM-1640 containing 10% FBS, and the periocular muscles and fascia were removed with eye scissors under a microscope. A circular incision was made along the edge of the cornea and sclera to separate the anterior and posterior chamber tissues. The infiltrating cells in the vitreous cavity were gently aspirated with a 200 μL pipette tip, and the lens, retina, and choroid were removed. The iris was separated from the ciliary body-corneal complex with forceps, gently torn into small pieces, and the culture medium was transferred to 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, incubate 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. The tissue was gently blown with a 1 mL pipette tip and passed through a 70 μm and 40 μm cell sieve in turn.

(2) Flow cytometry: The single-cell suspension prepared above was washed twice with PBS containing 1% BSA, incubated with CD16/32 antibody (1:200) on ice for 20 min, and then incubated with the target antibody on ice for 30 min. Protected from light, the suspension was 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 analyzed using flow cytometry. Data were analyzed using flowJo software.

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

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

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

The results of immunofluorescence staining of eyeball sections from mice in the AAV-VEGF-B and AAV-GFP groups are shown in Figures 6G (scale bar: 50 μm) and H. As shown in Figures 6G and H, the number of CD45-positive cells infiltrating the anterior chamber of mice in the AAV-VEGF-B group was significantly lower than that in the AAV-GFP group. This suggests that VEGF-B protein can effectively inhibit the infiltration of inflammatory cells in the eye and protect the integrity of the blood-aqueous humor barrier, indicating that VEGF-B protein has therapeutic effects on acute anterior uveitis.

The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents, but these modifications or replacements are all within the scope of protection of the present invention.

Claims (8)

Use of VEGF-B protein or an active peptide segment of VEGF-B protein in the preparation of a drug for preventing and/or treating acute anterior uveitis; the active peptide segment of VEGF-B protein comprises at least one of Peptide 1, Peptide 2, Peptide 3, Peptide 4, and Peptide 5; The amino acid sequence of Peptide 1 is shown in SEQ ID NO. 1; The amino acid sequence of Peptide 2 is shown in SEQ ID NO. 2. The amino acid sequence of Peptide 3 is shown in SEQ ID NO. 3. The amino acid sequence of Peptide 4 is shown in SEQ ID NO. 4. The amino acid sequence of Peptide5 is shown in SEQ ID NO.5. The use according to claim 1, wherein the drug for preventing and/or treating acute anterior uveitis is a drug for reducing ocular inflammatory cells. The use according to claim 2, wherein the inflammatory cells include CD45 positive cells. The use according to claim 1, characterized in that the active peptide segment of VEGF-B protein is Peptide 5. 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; the active peptide segment of VEGF-B protein comprises at least one of Peptide 1, Peptide 2, Peptide 3, Peptide 4, and Peptide 5; The amino acid sequence of Peptide 1 is shown in SEQ ID NO. 1; The amino acid sequence of Peptide 2 is shown in SEQ ID NO. 2. The amino acid sequence of Peptide 3 is shown in SEQ ID NO. 3. The amino acid sequence of Peptide 4 is shown in SEQ ID NO. 4. The amino acid sequence of Peptide5 is shown in SEQ ID NO.5. The use according to claim 5, characterized in that the drug for inhibiting macrophage M1 polarization is a drug for reducing the expression of inflammatory factors. The use according to claim 6, characterized in that the inflammatory factor includes at least one of TNF-α, IL-6, and IL-1β. A drug, characterized in that it contains a VEGF-B protein active peptide segment as an active ingredient; the VEGF-B protein active peptide segment 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 in SEQ ID NO. 1; The amino acid sequence of Peptide 2 is shown in SEQ ID NO. 2. The amino acid sequence of Peptide 3 is shown in SEQ ID NO. 3. The amino acid sequence of Peptide 4 is shown in SEQ ID NO. 4. The amino acid sequence of Peptide5 is shown in SEQ ID NO.5.