Use of primary urinary proteins to promote wound healing and reduce inflammation

Abstract

The application discloses application of main urinary protein in promoting wound healing and reducing inflammation. Through in-vivo animal and in-vitro cell experiments, it is found that the main urinary protein can promote angiogenesis, significantly shorten the wound healing time, and reduce inflammation. The experimental results show that the main urinary protein has the prospect of being used as a pharmaceutical active ingredient for related clinical application.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to the application of MUPs in the preparation of drugs that promote wound healing and alleviate inflammation-related diseases. Background Technology

[0002] Angiogenesis is the biological process by which new blood vessels grow on existing vascular structures, resulting from cell-cell, cell-matrix, and cell-cytokine interactions. Angiogenesis promotes wound healing and has significant physiological importance. Furthermore, inflammation is the pathological basis and clinical manifestation of many diseases; therefore, finding drugs that can improve inflammation provides new solutions and strategies for the prevention and treatment of numerous diseases.

[0003] Major urinary proteins (MUPs) are a class of lipid transport proteins capable of binding small molecules. Chinese patent CN113616777A indicates that MUP1 is an inhibitor of hepatic endoplasmic reticulum stress and has the effect of improving insulin resistance and treating diabetes. Other studies have found that the expression levels of platelet factor 4 (PF4) and MUPs are increased in the serum of rats stimulated with LPS, and that reducing PF4 produced during the inflammatory response can reduce the hypercoagulable state caused by the inflammatory response (“Screening of serum proteins affected by propofol in rats with endotoxemia using MALDI-TOF-MS technology”, Southern Medical University, 2010). However, there are currently no reports on the effects of MUPs themselves on accelerating angiogenesis, promoting wound healing, or inhibiting inflammatory responses. Summary of the Invention

[0004] The purpose of this invention is to provide applications for promoting wound healing and reducing inflammation by addressing major urinary proteins.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] The application of major urinary protein in the preparation of drugs for promoting wound healing.

[0007] Preferably, the major urinary protein can promote angiogenesis.

[0008] Preferably, the mechanism of action of the drug for promoting wound healing is to increase the expression level of vascular endothelial growth factor through intervention of major urinary proteins, thereby accelerating tissue angiogenesis and achieving the effect of promoting wound healing.

[0009] Preferably, the mechanism of action of the drug for promoting wound healing includes regulating the gene and / or protein levels of the wound tissue by increasing the level of major urinary proteins in the wound tissue.

[0010] Preferably, the drug for promoting wound healing is made from major urinary proteins (such as MUP15) and pharmaceutical excipients.

[0011] The application of major urinary protein in the preparation of drugs for reducing inflammation.

[0012] Preferably, the major urinary protein can reduce inflammation at the site of injury or in the vascular endothelium.

[0013] Preferably, the mechanism of action of the drug for reducing inflammation is to inhibit the activation of the NLRP3 inflammasome through intervention of major urinary proteins, and further reduce the levels of related inflammatory factors such as interleukin-18 and interleukin-1β, thereby inhibiting the inflammatory response.

[0014] Preferably, the mechanism of action of the drug for reducing inflammation includes regulating inflammatory factors in the injured area or vascular endothelium by increasing the level of major urinary proteins in the local area of ​​injury or the circulatory system.

[0015] Preferably, the medicament for reducing inflammation is made from major urinary proteins (such as MUP15) and pharmaceutical excipients.

[0016] The beneficial effects of this invention are reflected in:

[0017] This invention, based on the significant role of major urinary proteins in promoting local tissue angiogenesis and their remarkable effect in shortening wound healing time, demonstrates that major urinary proteins (such as MUP15) can be used in the preparation of drugs that promote wound healing. Furthermore, this invention utilizes an inflammation model to demonstrate that major urinary proteins (such as MUP15) have a significant inhibitory effect on inflammatory responses, thus enabling their use in the preparation of drugs that alleviate inflammation. Based on the use of major urinary proteins as an effective intervention in the preparation of drugs that promote wound healing and reduce inflammation, this invention also proposes that major urinary proteins can be used in the preparation of drugs for the prevention and treatment of ischemic diseases. Attached Figure Description

[0018] Figure 1 A gel image for MUP15 protein detection.

[0019] Figure 2 Gel image of MUP15 protein purification.

[0020] Figure 3 The in vivo animal experiment results on the wound-promoting effect of MUP15: A. Gross images of wound healing in two groups of mice; B. Statistical graph of wound area change, n=3. * P<0.05, control group vs. treatment group, wound area change = (wound area on the previous day - wound area on the next day) / wound area on the previous day × 100%.

[0021] Figure 4 Results of in vivo animal experiments on MUP15 promoting tissue angiogenesis: A. Schematic diagram of the subdermal implantation model (images taken out and recorded after coagulation); B. Gross images comparing different groups.

[0022] Figure 5 In vitro experimental validation of the effects of MUP15 on endothelial cell proliferation, protein expression, migration, tube formation, and budding: A. CCK8 assay to detect the effect of MUP15 on endothelial cell proliferation, n=6, * P<0.05, ** P<0.01, *** P < 0.001, compared with the control group; B.WB (Western Blot) was used to detect the effect of MUP15 on the expression level of endothelial cell proliferation-related proteins, n = 3. * P<0.05, ** P<0.01, *** P<0.001, compared with the control group; C. Scratch assay to detect the effect of MUP15 on endothelial cell migration ability, n=8, ** P<0.01, compared with the control group, cell migration rate = (0-hour scratch area - 12-hour scratch area) / 0-hour scratch area × 100%; D. Tube formation assay to detect the effect of MUP15 on the tube-forming ability of endothelial cells, n=8, * P<0.05, compared with the control group; E. Sprouting experiment to detect the effect of MUP15 on the vascular budding ability of vascular rings, n=6, ** P<0.01, compared with the control group.

[0023] Figure 6 In vivo animal experiments demonstrating the inhibitory effect of MUP15 on monosodium urate (MSU)-induced knee osteoarthritis: A. Changes in joint width over 24 hours; B. IL-1β expression level; C. IL-18 expression level; n=6, * P<0.05, ** P<0.01, *** P<0.001. Detailed Implementation

[0024] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. These embodiments are illustrative of the invention and are not intended to limit the scope of protection of the invention.

[0025] (I) Preparation of MUP protein

[0026] By comparing all 22 MUP proteins (MUP1, MUP2, MUP3, MUP4, MUP5, MUP6, MUP7, MUP8, MUP9, MUP10, MUP11, MUP12, MUP13, MUP14, MUP15, MUP16, MUP17, MUP18, MUP19, MUP20, MUP21, and MUP22) from the mouse MUP family, the results showed that the sequences of different MUP proteins were highly conserved. Therefore, one MUP protein (specifically MUP15) was selected as the target protein for expression experiments, and the resulting target protein expression product was identified. Subsequent experiments were then conducted using the MUP protein obtained through expression.

[0027] 1.1 Proteins of the MUPs family

[0028] MUP1:

[0029] MKMLLLLCLGLTLVCVHAEEASSTGRNFNVEKINGEWHTIILASDKREKIEDNGNFRLFLEQIHVLENSLVLKFHTVRDEECSELSMVADKTEKAGEYSVTYDGFNTFTIPKTDYDNFLMAHLINEKDGETFQLMGLYGREPDLSSDIKERFAQLCEKHGILRENIIDLSNANRCLQARE

[0030] MUP2:

[0031] MKMLLLLCLGLTLVCVHAEEASSTGRNFNVEKINGEWHTIILASDKREKIEDNGNFRLFLEQIHVLEKSLVLKFHTVRDEECSELSMVADKTEKAGEYSVTYDGFNTFTIPKTDYDNFLMAHLINEKDGETFQLMGLYGREPDLSSDIKERFAKLCEEHGILRENIIDLSNANRCLQARE

[0032] MUP3:

[0033] MKLLLPLLLLLCLELTLVCIHAEESSSMERNFNVEQISGYWFSIAEASYEREKIEEHGSMRAFVENITVLENSLVFKFHLIVNEECTEMTAIGEQTEKAGIYYMNYDGFNTFSILKTDYDNYIMIHLINKKDGKTFQLMELYGREPDLSLDIKEKFAKLCEEHGIIRENIIDLTNVNRCLEARE

[0034] MUP4:

[0035] MKLLLCLGLTLVCIHAEEATSKGQNLNVEKINGEWFSILLASDKREKIEEHGSMRVFVEHIHVLENSLAFKFHTVIDGECSEIFLVADKTEKAGEYSVMYDGFNTFTILKTDYDNYIMFHLINEKDGKTFQLMELYGRKADLNSDIKEKFVKLCEEHGIIKENIIDLTKTNRCLKARE

[0036] MUP5:

[0037] MKLLLLLCLELTLVYVHAEEASSEGQNLNVEKINGKWFSILLASDKREKIEEHGTMRVFVEHIDVLENSLAFKFHTVIDEECTEIYLVADKTEKAGEYSVTYDGFNTFTILKTDYDNYIMFHLINKKDEENFQLMELFGREPDLSSDIKEKFAKLCEEHGIVRENIIDLSNANRCLQARE

[0038] MUP6:

[0039] MKMLLLLCLGLTLVCVHAEEASSTGRNFNVEKINGEWHTIILASDKREKIEDNGNFRLFLEQIHVLENSLVLKFHTVRDEECSELSMVADKTEKAGEYSVTYDGFNTFTIPKTDYDNFLMAHLINEKDGETFQLMGLYGREPDLSSDIKERFAQLCEEHGILRENIIDLSNANRCLQARE

[0040] MUP7:

[0041] MKMLLLLCLGLTLVCVHAEEASSTGRNFNVEKINGEWHTIILASDKREKIEDNGNFRLFLEQIHVLENSLVLKVHTVRDEECSELSMVADKTEKAGEYSVTYDGFNTFTIPKTDYDNFLMAHLINEKDGETFQLMGLYGREPDLSSDIKERFAQLCEKHGILRENIIDLSNAMDLVPEHVLVLTLQIAASRPENEEWPEPPVLSGDFSPGLHHHPFLSIQHPQYKFCDLHSILSHMUP8:

[0042] MKMLLLLCVGLTLVCVHAEEASSTGRNFNVEKINGEWHTIILASDKREKIEDNGNFRLFLEQIRVLENSLVLKVHTVRDEECSELSMVADKTEKAGEYSVTYDGFNTFTIPKTDYDNFLMAHLINEKDGETFQLMGLYGREPDLSSDIKERFAQLCEEHGILRENIIDLSNAMDLVPEHVLVLTRQIAASRPENEEWPEPPVLSGDFSPGLHHHPFLSIQHPQYKFCDLHSILSHMUP9:

[0043] MKMLLLLLCLGLTLVCVHAEEASSTGRNFNVEKINGEWHTIILASDKREKIEDNGNFRLFLEQIHVLENSLVLKFHTVRDEECSELSMVADKTEKAGEYSVTYDGFNTFTIPKTDYDNFLMAHLINEKDGETFQLMGLYGREPDLSSDIKERFAQLCEEHGILRENIIDLSNANRCLQARE

[0044] MUP10:

[0045] MKMMLLLCLGLTLVCVHAEEASSTGRNFNVEKINGEWHTIILASDKREKIEDNGNFRLFLEQIHVLEKSLVLKFHTVRDEECSELSMVADKTEKAGEYSVTYDGFNTFTIPKTDYDNFLMAHLINEKDGETFQLMGLYGREPDLSSDIKERFAQLCEEHGILRENIIDLSNANRCLQARE

[0046] MUP11:

[0047] MKMLLLLLCLGLTLVCVHAEEASSTGRNFNVEKINGEWHTIILASDKREKIEDNGNFRLFLEQIHVLENSLVLKFHTVRDEECSELSMVADKTEKAGEYSVTYDGFNTFTIPKTDYDNFLMAHLINEKDGETFQLMGLYGREPDLSSDIKERFAQLCEEHGILRENIIDLSNANRCLQARE

[0048] MUP12:

[0049] MKMLLLLCLGLTLVCVHAEEASSTGRNFNVEKINGEWHTIILASDKREKIEDNGNFRLFLEQIRVLENSLVLKFHTVRDEECSELSMVADKTEKAGEYSVTYDGFNTFTIPKTDYDNFLMAHLINEKDGETFQLMGLYGREPDLSSDIKERFAQLCEEHGILRENIIDLSNANRCLQARE

[0050] MUP13:

[0051] MKMLLLLCLGLTLVCVHAEEASSTGRNFNVEKINGEWHTIILASDKREKIEEHGNFRLFLEQIHVLENSLVLKVHTVRDEECSELSMVADKTEKAGKYSVTYDGFNTFTIPKTDYDNFLMAHLINEKDGETFQLMGLYGREPDLSSDIKERFAQLCEEHGILRENIIDLSNAMDLVPEHVLVLTLQIAASRPENEEWPEPPVLSGDFSPGLHHHPFLSIQHPQYKFCDLHSILSHMUP14:

[0052] MKMLLLLCLGLTLVCVHAEEASSTGRNFNVEKINGEWHTIILASDKREKIEDNGNFRLFLEQIRVLENSLVLKFHTVRDEECSELSMVADKTEKAGEYSVTYDGFNTFTIPKTDYDNFLMAHLINEKDGETFQLMGLYGREPDLSSDIKERFAQLCEEHGILRENIIDLSNANRCLQARE

[0053] MUP15:

[0054] MKMLLLLCLGLTLVCVHAEEASSTGRNFNVQKINGEWHTIILASDKREKIEDNGNFRLFLEQIHVLENSLVLKFHTVRDEECSELSMVADKTEKAGEYSVTYDGFNTFTIPKTDYDNFLMAHLINEKDGETFQLMGLYGREPDLSSDIKERFAQLCEKHGILRENIIDLSNANRCLQARE

[0055] MUP16:

[0056] MKMLLLLLCLGLTLVCVHAEEASSTGRNFNVEKINGEWHTIILASDKREKIEDNGNFRLFLEQIHVLENSLVLKFHTVRDEECSELSMVADKTEKAGEYSVTYDGFNTFTIPKTDYDNFLMAHLINEKDGETFQLMGLYGREPDLSSDIKERFAQLCEEHGILRENIIDLSNANRCLQARE

[0057] MUP17:

[0058] MKMLLLLCLGLTLVCVHAEEASSTGRNFNVEKINGEWHTIILASDKREKIEEHGNFRLFLEQIHVLENSLVLKVHTVRDEECSELSMVADKTEKAGEYSVTYDGFNTFTIPKTDYDNFLMAHLINEKDGETFQLMGLYGREPDLSSDIKERFAQLCEEHGILRENIIDLSNANRCLQARE

[0059] MUP18:

[0060] MKMLLLLLCLGLTLVCVHAEEASSTGRNFNVEKINGEWHTIILASDKREKIEDNGNFRLFLEQIHVLENSLVLKFHTVRDEECSELSMVADKTEKAGEYSVTYDGFNTFTIPKTDYDNFLMAHLINEKDGETFQLMGLYGREPDLSSDIKERFAQLCEEHGILRENIIDLSNANRCLQARE

[0061] MUP19:

[0062] MKMLLLLCLGLTLVCVHAEEASSTGRNFNVEKINGEWHTIILASDKREKIEDNGNFRLFLEQIHVLENSLVLKFHTVRDEECSELSMVADKTEKAGEYSVTYDGFNTFTIPKTDYDNFLMAHLINEKDGETFQLMGLYGREPDLSSDIKERFAQLCEEHGILRENIIDLSNASKSGFSYFSHPEYLS

[0063] MUP20:

[0064] MKLLVLLLCLGLTLVCVHAEEASSMERNFNVEKINGEWYTIMLATDKREKIEEHGSMRVFVEYIHVLENSLALKFHIIINEECSEIFLVADKTEKAGEYSVTYDGSNTFTILKTDYDNYIMIHLINKKDGETFQLMELYGREPDLSSDIKEKFAQLSEEHGIVRENIIDLTNANRCLEARE

[0065] MUP21:

[0066] MKLLLLLLCLGLTIVCIQAEEYSSMGRNFNVEQISGYWFSIAEASDEREKIEEHGSMRAFVENITVLENSLVFKFHFIVNEECTEMTLIGEETEKAGIYYLNYDGFNTFTILKTDYDNYIMIYLINEKDGETFQLMELYGREPYLSLDIKEKFAKLCEEHGIIRENIIDLTNVNRCLEARE

[0067] MUP22:

[0068] MKMLLLLLCLGLTLVCVHAEEASSTGRNFNVEKINGEWHTIILASDKREKIEDNGNFRLFLEQIHVLEKSLVLKFHTVRDEECSELSMVADKTEKAGEYSVTYDGFNTFTIPKTDYDNFLMAHLINEKDGETFQLMGLYGREPDLSSDIKERFAQLCEEHGILRENIIDLSNARCLQARE

[0069] 1.2 MUP protein expression and separation identification

[0070] (1) MUP15 protein expression vector source: synthesized by ABclonal.

[0071] (2) Identification of Ligation Products: HEK293 cells were transfected for protein expression. Before transfection, the viable cell density was approximately 2.5 × 10^6 cells / mL, and after transfection, the cell viability was maintained above 95%. After 24 hours, feed was added to the culture medium for continued culture. The cultured cells were collected after 3–4 days. The collected cell supernatant (the supernatant obtained after centrifuging cells and culture medium together) was analyzed by Western blotting (WB) to determine the expression status of the target protein. The WB detection conditions and electrophoresis results are as follows: Figure 1 As shown (band size 18.6 kDa), the expressed MUP15 is a his-tagged protein, which was detected using the corresponding his-tagged antibody.

[0072] (3) Separation and purification of the expression products using Ni columns:

[0073] The matrix (packing material) was equilibrated with an imidazole-containing buffer, and cell supernatant was added for incubation. After incubation, elution was performed to remove non-specifically bound proteins from the Ni column. The target protein MUP15 was then eluted using a multi-gradient imidazole solution. The target protein was ultimately eluted in imidazole solutions of 40 mM, 80 mM, 250 mM, and 500 mM. Figure 2 As shown (top: cell supernatant; FT, W: <40mM low concentration imidazole to remove non-specifically bound proteins; -1: first elution with the corresponding concentration of imidazole; -2: second elution with the corresponding concentration of imidazole; matrix: after eluting the target protein with all imidazole solutions to ensure that all target proteins have been eluted). The target protein with high purity obtained by elution was dialyzed and concentrated using a PBS buffer system at pH 7.4. The concentration of MUP15 protein solution was then measured, and the endotoxin content of MUP15 protein solution was confirmed to be <0.1EU / μg using the Limulus Amebocyte Lysate (LAL) assay. At this point, the MUP protein used in subsequent experiments was obtained.

[0074] (II) MUPs Intervention Experiment

[0075] 2.1 MUPs accelerate wound healing in mice

[0076] Eight-week-old male C57BL / 6J mice were randomly divided into two groups: a control group and a treatment group. Mice were anesthetized with sodium pentobarbital (0.5%). After hair removal from the back, two incisions were made on both sides of the spine using a 6mm skin puncture device. The mice were then carefully monitored until they awoke and were then fed normally. During wound healing, 0.25 mg / kg body weight of MUP protein (dissolved and diluted in 0.2 mL of physiological saline) was injected every odd-numbered day at four points along the edge of each wound. The wounds were photographed every other day. Figure 3 A) The wound area size is quantified and the wound area change is calculated using ImageJ.

[0077] By statistically analyzing the changes in wound size in each mouse in the control group (saline) and the treatment group (MUP protein), it was found that the wound healing rate in the treatment group was faster than that in the control group, and a statistically significant difference appeared on the fifth day (P<0.05). Figure 3 B). The results showed that administration of MUP protein significantly shortened the wound healing time in mice, suggesting that MUPs can promote wound healing in mice.

[0078] 2.2 MUPs increase intramacrogel vessel density

[0079] Eight-week-old male C57BL / 6J mice were randomly divided into two groups: an experimental group and a control group. Mice were anesthetized with sodium pentobarbital (1%), fixed in a supine position on a board with their tails facing the operator, and the hind limbs were disinfected. An equal volume of physiological saline (control group) or MUP protein dissolved in physiological saline (experimental group, 0.5 mg / kg) was mixed with matrix gel. Then, 0.5 mL of the diluted matrix gel, which had been mixed in an ice-water bath, was rapidly injected subcutaneously into the groin area of ​​the mice using a 1 mL syringe. The mice were kept fixed for approximately 30 minutes to ensure the matrix gel had completely solidified into a gel. Figure 4 A), and then carefully cared for until the mouse woke up. Afterwards, it was fed normally, and a week later, the diluted matrix gel was removed, and a rough image of the gel was recorded using a digital camera. Figure 4 B). The results showed that the blood vessel density in the matrix gel mixed with MUP protein was significantly increased (n=3, P<0.01, control group vs. experimental group), suggesting that MUPs can promote angiogenesis.

[0080] 2.3 Effects of MUPs on endothelial cell proliferation, protein expression, migration, tube formation, and budding processes

[0081] (1) Human aortic endothelial cells (purchased from Shanghai Baili Biotechnology Co., Ltd.) were seeded in 96-well plates at a density of 0.4 × 10⁻⁶. 4 Cells were cultured in 96-well plates for 24 hours, and then 10 μL of CCK-8 was added to each well for 1 hour of incubation. Cell counts were then determined using a microplate reader (490 nm) and calculated based on a standard curve. Results showed that compared to the control group (no MUP protein treatment), MUP protein significantly increased the proliferation rate of endothelial cells. Figure 5 A), and the non-toxic concentration range is 0.001 μg / mL to 10 μg / mL.

[0082] (2) Human aortic endothelial cells were treated with MUP protein at concentrations of 0.05 μg / mL, 0.1 μg / mL, and 0.5 μg / mL, respectively. After treatment, the culture medium was removed, and the cells were washed twice with PBS. RIPA lysis buffer containing protease inhibitors was added, and the cells were scraped off with a cell scraper and aspirated into centrifuge tubes. The centrifuge tubes were shaken, placed on ice for 10 minutes, shaken again, and centrifuged at 12,000 rpm for 15 minutes at 4°C. The supernatant was collected, BCA quantification was performed, and the mixture was leveled. 5× Loading Buffer and RIPA lysis buffer were added to adjust the protein concentration of each group to the same level. The mixture was then boiled in a metal bath at 100°C for 10 minutes. Subsequently, the sample (20 μg) was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the proteins were transferred to a polyvinylidene fluoride (PVDF) membrane. The transferred membrane was incubated with primary antibody, and then further incubated at room temperature with horseradish peroxide-labeled secondary antibody diluted in 5% skim milk. Finally, ECL chromogenic buffer was used to visualize protein bands, and quantification was performed using densitometric analysis with Quantity One software. Experimental results ( Figure 5 B) shows that, compared with the control group (without MUP protein treatment), MUP protein significantly increased the expression levels of vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor 2 (VEGFR2), and phosphorylated vascular endothelial growth factor receptor 2 (p-VEGFR2). These results suggest that MUPs can promote VEGF expression in endothelial cells and enhance cell proliferation signaling.

[0083] (3) Human aortic endothelial cells were seeded into 6-well plates with horizontal streaks at the bottom. After the cell monolayers reached confluence, vertical streaks were made in the cell plates using a 200 μL pipette tip. At 0 h, MUP protein (0.1 μg / mL, experimental group) or no MUP protein was administered to the culture medium (control group). The width of the streaks at 0 h was recorded using a bright-field inverted microscope. The closure of the streaks was observed and photographed over the next 12 h. The results showed that MUP protein increased the migration ability of endothelial cells in the experimental group (P<0.01). Figure 5 C); A 1:1 dilution of matrix gel and ECs medium containing 5% FBS and 100 μg / mL growth supplement was performed. 50 μL of the diluted matrix gel was added to each well of a 96-well plate using a pre-cooled pipette tip. The plate was then incubated at 37°C for 30 min to allow the matrix gel to solidify. Endothelial cells pretreated with MUP protein (0.1 μg / mL) (experimental group) or untreated (control group) were digested with trypsin, centrifuged, and resuspended. 10,000 cells were added to each well. After culturing for 4 h, tubule formation within the matrix gel was observed using a bright-field inverted microscope. The results showed that MUP protein increased the tubule-forming ability of endothelial cells in the experimental group (P<0.05). Figure 5D); After rats were euthanized by intraperitoneal injection of sodium pentobarbital, the thoracic aorta was dissected and separated. The surrounding fat and other connective tissue were removed, and the aorta was cut into vascular rings approximately 0.5 mm wide. These rings were placed in a culture medium containing MUP protein (0.1 μg / mL, experimental group) or without MUP protein (control group) for 24 hours. Each aortic vascular ring was embedded in a cured matrix gel and then covered with 30 μL of matrix gel. Vessel budding was stimulated by supplementing the ECs culture medium with 5% FBS and 100 μg / mL growth supplement. The culture medium was changed every other day. After 4 days of culture, images of vascular budding were obtained using a bright-field inverted microscope. The results showed that MUP protein increased the budding ability of endothelial cells in the experimental group (P<0.01). Figure 5 E). These results suggest that upregulating MUPs can significantly enhance angiogenesis.

[0084] The results above suggest that MUPs can significantly promote angiogenesis in endothelial cells.

[0085] 2.4 MUPs have an inhibitory effect on monosodium urate (MSU)-induced knee osteoarthritis.

[0086] MUP protein (0.13 mg / kg or 1.3 mg / kg) was injected into the joint cavity of the left knee of mice for pre-protection (control group received PBS). After 30 minutes, MSU was injected into the joint cavity of the left knee to induce knee arthritis (control group received saline). The width of the left knee joint was measured at 1 h, 3 h, 6 h, 12 h, and 24 h. Figure 6 A), mice were euthanized by cervical dislocation 24 hours later, and the left knee joint was removed and placed in a 12-well plate containing 200 μL of opti-mem (containing 1% penicillin antibody) for 1 hour. The supernatant was collected and IL-18 was detected. Figure 6 C), IL-1β Figure 6 B). The results showed that MUP protein could inhibit MSU-induced knee arthritis, suggesting that MUPs have anti-inflammatory effects and may help reduce the occurrence of inflammation-related diseases.

[0087] In summary, this invention experimentally investigated the effects of MUPs on accelerating angiogenesis and promoting wound healing, as well as their effects on reducing inflammation. The experimental results indicate that MUPs have the potential for clinical application as active pharmaceutical ingredients.

Claims

1. Application of major urinary protein MUP15 in the preparation of drugs for promoting wound healing.

2. The application as described in claim 1, characterized in that: The main urinary protein MUP15 promotes angiogenesis.

3. The application as described in claim 2, characterized in that: The major urinary protein MUP15 accelerates tissue angiogenesis by increasing the expression level of vascular endothelial growth factor.

4. The application as described in claim 1, characterized in that: The drug works by increasing the level of MUP15, the main urinary protein in wound tissue.

5. The application as described in claim 1, characterized in that: The drug is made from the main urinary protein MUP15 and excipients.

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