Application of pancreatitis related protein or derivative thereof in preparation of medicine for treating suppurative arthritis

Abstract

The invention discloses application of pancreatitis-related protein or a derivative thereof in preparation of a medicine for treating suppurative arthritis. According to the present invention, the pancreatitis-associated protein can effectively inhibit the growth of suppurative bacteria in articular cavities and relieve the excessive inflammatory response of joint parts so as to protect articular cartilage, and the pancreatitis-associated protein provides the excellent treatment effect on suppurative arthritis compared with the positive control drug vancomycin, the compound can be used for preparing medicines for treating suppurative arthritis and has an important application prospect.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and relates to the application of pancreatitis associated protein (PAP) or its derivatives in the preparation of a drug for treating suppurative arthritis. Background Art

[0002] Suppurative arthritis is an acute infectious disease in which purulent bacteria directly infect synovial joints, leading to inflammatory damage to joint tissues and even loss of function. It is common in people with low immune function, such as the elderly, children and patients with chronic joint diseases. Hematogenous infection is the main cause of the disease. In addition, bacteria can directly invade joint tissues through joint puncture, surgery, injury or infection of adjacent joint tissues. In recent years, with the increase in the frequency of invasive orthopedic surgery and immunotherapy, the incidence of suppurative arthritis has increased year by year, and the increase in recurrence rate and mortality caused by improper infection control has seriously affected the quality of life of patients and endangered their health and safety. The main pathogen of suppurative arthritis is Staphylococcus aureus (S. aureus), but in the past decade, with the emergence and rapid spread of antibiotic-resistant strains, methicillin-resistant Staphylococcus aureus (MRSA) infection has become the main pathogen of suppurative arthritis, bringing greater difficulty and challenges to treatment.

[0003] At present, the main treatment options for suppurative arthritis are drug therapy and surgical treatment. Drug therapy refers to the extraction of joint effusion and injection of antibiotics into the joint cavity under the premise of sufficient systemic use of antibiotics. This treatment option is only suitable for the early stage of suppurative arthritis, and there may be a risk of treatment failure due to incomplete bacterial removal. In addition, the early stage of suppurative arthritis (i.e., serous exudation stage) is difficult to define, which also leads to difficulties in the implementation of drug treatment options. At present, surgical treatment is clinically preferred. Through open wounds or arthroscopy, the joint cavity infection site is intervened, joint pus is thoroughly extracted, and it is rinsed with saline, and then an appropriate amount of antibiotics is injected for adjuvant treatment. In the short term, repeated debridement may be required, and the treatment frequency is high. In addition, the affected limb needs to be fixed and properly pulled to prevent the spread of infection and maintain functional position to prevent joint deformity. Although surgical intervention can completely remove the lesions, it has high requirements for surgical techniques and there is a risk of incomplete surgery. At the same time, the physical and mental discomfort caused by frequent surgery and postoperative care also seriously affect the quality of life of patients. In general, whether it is drug treatment or surgical treatment, it is inseparable from the use of antibacterial drugs. On the one hand, they can completely eliminate pathogens, and on the other hand, they can prevent secondary infection of tissues.

[0004] Antimicrobial drugs refer to drugs with antibacterial or bactericidal activity, mainly including natural antibiotics and synthetic antimicrobial drugs, in addition to secondary metabolites (such as antimicrobial peptides) obtained by culturing bacteria, actinomycetes, fungi and other microorganisms. MRSA can produce a large amount of β-lactamase, so it is resistant to other β-lactam and cephalosporin antibiotics with similar structures to methicillin, but is sensitive to vancomycin. Therefore, vancomycin is the first choice for the treatment of suppurative arthritis caused by MRSA infection in clinical practice. However, long-term excessive use of antibiotics is the most direct cause of bacterial resistance. In recent years, studies have shown that the minimum inhibitory concentration (MIC) value of vancomycin for MRSA has an upward trend. Some European countries emphasize in the guidelines for the treatment of MRSA infections that vancomycin should be limited to severe MRSA infections. In addition to vancomycin, linezolid, as a synthetic oxazolidinone antibiotic, can also be used to treat MRSA infections, showing good antibacterial effects, and can even be used to further treat vancomycin-resistant MRSA infections. Although linezolid is not easy to induce bacterial resistance, its treatment is often accompanied by adverse reactions such as diarrhea, headache and nausea. As the treatment time goes by, it may even cause systemic toxicity and side effects such as bone marrow suppression, blindness and lactic acidosis. Therefore, it is urgent to screen and develop safe and effective antibacterial drugs for the treatment of MRSA infection.

[0005] In addition to using antimicrobial drugs to inhibit the reproduction of pathogens, arthritis damage and cartilage lesions caused by infection are also difficult to treat suppurative arthritis, which requires further combined treatment with anti-inflammatory drugs and chondroprotective agents. Anti-inflammatory drugs are drugs used to treat reactive inflammation after tissue damage, and are divided into two categories: steroidal anti-inflammatory drugs and non-steroidal anti-inflammatory drugs. Steroidal anti-inflammatory drugs only play an anti-inflammatory role by inhibiting the synthesis of prostaglandins, inhibiting the aggregation of white blood cells, reducing the formation of bradykinin, and inhibiting platelet aggregation. Nonsteroidal anti-inflammatory drugs (NSAIDs) are a class of anti-inflammatory drugs that do not contain steroidal structures. They inhibit the activity of cyclooxygenase, thereby inhibiting the final generation of prostacyclin (PGI1), prostaglandins (PGE1, PGE2) and thromboxane A2 (TXA2) from arachidonic acid. They can also inhibit the release of bradykinin during inflammation, change lymphocyte response, reduce the migration and phagocytosis of granulocytes and monocytes, and have anti-inflammatory and analgesic effects. They are widely used in clinical practice to relieve osteoarthritis, rheumatoid arthritis, multiple fevers and various pain symptoms. Therefore, nonsteroidal anti-inflammatory drugs such as ibuprofen, acetaminophen or diclofenac sodium are usually used in combination with antibiotics to treat suppurative arthritis, but they are limited to relieving pain and mild inflammation. Long-term use will have adverse toxic and side effects on the gastrointestinal tract and liver and kidney functions of the human body, and symptoms are prone to recurrence after discontinuation of the drug. In addition, repeated or superimposed use of similar drugs will not only not increase the efficacy, but will increase the incidence of adverse reactions. In the existing treatment options, analgesic treatments cannot inhibit further deterioration of joint structure, and chondroitin sulfates need to be combined to relieve joint damage and inhibit further joint degeneration. Chondroitin sulfates are a type of glycosaminoglycan drug, mainly including hyaluronic acid, glucosamine and chondroitin sulfate. Although glucosamine and chondroitin sulfate can theoretically protect or even repair cartilage by stimulating the synthesis of new cartilage matrix, and their combined use is also the most common clinical treatment option, there is no sufficient evidence to confirm their therapeutic effects. The 2011 European guidelines clearly pointed out that glucosamine and chondroitin sulfate may have a weak therapeutic effect on osteoarthritis, but it still needs to be further confirmed by new studies. For septic arthritis, an acute disease that can cause joint destruction and loss of function, in order to prevent joint deformity, the joints should be treated with traction fixation and movement should be reduced or even stopped. The lubricating and protective effect of chondroitin sulfates is no longer important, so glucosamine and chondroitin sulfate can only be used as nutritional supplements. In addition, the combined use of the above-mentioned drugs for inflammation and tissue damage repair will increase the burden on the patient's liver and kidneys, and the overly complicated medication regimen also brings inconvenience to actual treatment.

[0006] Antibacterial peptides (ABPs) are polypeptides with broad-spectrum and high-efficiency bactericidal activity against bacteria. They originally refer to a class of alkaline polypeptide substances with antibacterial activity that are induced in insects. They are composed of 20 to 60 amino acid residues and have a molecular weight of about 2000 to 7000. Initially, Swedish scientist G. Boman and others discovered that when the diapause pupae of North American silkworms were induced by external stimuli, polypeptide substances with antibacterial effects were produced in their hemolymph. This type of antibacterial polypeptide was named Cecropins. Later, antibacterial polypeptides with similar structures were also isolated from other insects, amphibians, and mammals. ABPs act on the bacterial cell membrane to form transmembrane ion channels on the membrane, destroying the integrity of the bacterial outer membrane. They have broad-spectrum and high-efficiency bactericidal activity, especially against some drug-resistant pathogens. Moreover, ABP has selective immune activation and regulation functions. Studies have shown that antimicrobial peptides can selectively stimulate the expression and release of inflammatory inhibitory cytokines and immune cell chemokines through the p38 MAPK signaling pathway without causing the activation of a large number of harmful inflammatory factors, and have a good preventive and protective effect on systemic and lethal sepsis. In addition, as a class of natural products, ABP has good biocompatibility and low target resistance. Therefore, as a new type of antibacterial drug, antimicrobial peptides have good application potential and value in the treatment of MRSA-induced suppurative arthritis in mice.

[0007] Pancreatitis associated proteins (PAP), also known as regenerative gene family proteins (REG), are a class of antibacterial proteins that are highly expressed under inflammatory conditions of the digestive tract. PAP proteins specifically target Gram-positive bacteria. Their antibacterial effect is mediated by the formation of hexameric membrane-permeable oligomeric pores by binding to the exposed carbohydrate part of the peptidoglycan surface to mediate bacterial death, which is consistent with the antibacterial mechanism that antimicrobial peptides aggregate on the bacterial surface to form permeable pores and mediate bacterial perforation and lysis. At the same time, PAP proteins can also exert the potential of regulating the liver-gut axis and the brain-gut axis by regulating intestinal flora, and show good therapeutic effects in the treatment of various diseases. In addition, PAP proteins are also involved in the repair and regeneration of tissue inflammatory damage. Its structure mainly contains the active site of the C-type lectin-like domain and a signal peptide, which can promote cell proliferation, resist cell apoptosis, regulate the expression of inflammatory factors, and maintain the homeostasis of glucose and lipid metabolism in the extracellular matrix through autocrine or paracrine forms. A large number of studies have shown that PAP protein plays a vital role in the repair process of various tissues after damage. For example, transplanting a small amount of hepatocytes from PAP-overexpressing transgenic mice into wild-type mice with hepatectomy can promote rapid regeneration of liver tissue; injecting a small dose of PAP protein after the mouse liver is removed can also obtain similar results, indicating that the repair of liver tissue is closely related to PAP protein. In addition, PAP protein can promote wound healing by promoting keratinocyte proliferation; PAP protein can also be activated after gastric mucosal injury, thereby promoting the healing of lesioned mucosa and preventing hepatocyte apoptosis in acute liver failure. It is worth noting that some studies have found that the expression of PAP protein increases abnormally after fracture, and then drops to the basal level six weeks after the fracture heals. Periosteal cells cultured in vitro do not express PAP, but the addition of IL-6 can significantly induce PAP expression. These results indicate that the IL-6 / PAP pathway is involved in the osteogenic response of the periosteum and promotes fracture repair. To date, there have been no reports of the use of PAP protein in the treatment of septic arthritis. Summary of the invention

[0008] The present invention provides an application of a pancreatitis associated protein (PAP) or a derivative thereof in preparing a medicine for treating suppurative arthritis.

[0009] The suppurative arthritis described in the present invention includes suppurative arthritis caused by Gram-positive bacteria infection such as Staphylococcus aureus and methicillin-resistant Staphylococcus aureus.

[0010] The PAP protein described in the present invention includes natural PAP protein and recombinant PAP protein constructed by genetic engineering.

[0011] Specifically, the PAP protein is naturally extracted PAP protein, such as natural PAP protein extracted from pancreatic juice, intestinal juice, bile, pancreatic tissue, intestinal tissue of mice, rats, guinea pigs, cows, pigs, dogs, rabbits and other animals, or natural PAP protein or recombinant PAP protein fermented by microorganisms.

[0012] The dosage form of the medicine for treating suppurative arthritis of the present invention is an injection dosage form.

[0013] The administration method of the drug for treating suppurative arthritis of the present invention is local injection, such as subcutaneous or intra-articular injection, or systemic injection for treating suppurative arthritis. In a specific embodiment of the present invention, the administration method adopted is local injection into the suppurative joint cavity.

[0014] The drug for treating suppurative arthritis of the present invention can be applied to any animal that may or has developed suppurative arthritis, including humans and non-human animals, such as pets or livestock.

[0015] The dosage of the drug for treating suppurative arthritis of the present invention depends on the age, health and weight of the recipient, the frequency of treatment, the route of administration, etc., and can be 100-1000 μg / kg. In a specific embodiment of the present invention, the dosage of the drug for treating suppurative arthritis is 180 μg / kg.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention discovered for the first time that PAP protein can inhibit suppurative bacterial infection in the joint cavity, relieve excessive inflammation in the joint area, promote chondrocyte proliferation, protect and / or repair articular cartilage, and can be prepared into a drug for the treatment of suppurative arthritis. Specifically, (1) the present invention confirmed that PAP protein has good antibacterial effect on MRSA and SA in vitro by respectively determining the minimum inhibitory concentration and minimum bactericidal concentration of PAP protein for MRSA and SA; (2) after administration of PAP protein, it was observed that RAW264.7 cell morphology had a depolarization effect of M1, and inflammatory factors (TNF-α, IFN-γ, IL-6) and anti-inflammatory factors (IL-4) in the cell culture medium were detected, confirming that PAP protein has an immunomodulatory effect on mouse-derived RAW264.7 macrophages in vitro; ( 3) A suppurative arthritis model was established in mice by intra-articular injection of MRSA, and PAP protein was injected into the knee joint cavity of mice for treatment. Compared with the positive drug (vancomycin) group, the PAP protein group eliminated joint swelling earlier than the positive drug group, and the plantar inkblot results showed that the PAP protein was basically consistent with the blank control group. However, although the joint swelling of the mice in the positive drug group was eliminated after two weeks of treatment, incomplete footprints, i.e., joint pain, were still present, confirming that PAP protein showed a superior therapeutic effect on suppurative arthritis compared with the positive control drug vancomycin. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The minimum inhibitory concentration (MIC) of PAP protein in Example 1 is shown in Figure 1; A is a microplate intuitive diagram, B is the coating of each group of S. aureus (SA) and MRSA in a 96-well plate, and C is the bacterial solution of each well at OD 600 The absorbance value at .

[0019] Figure 2 1 is a graph showing the results of the determination of the minimum bactericidal concentration (MBC) of the PAP protein in Example 1; wherein A is the coating of each group diluted to an effective counting multiple in the MIC, and B is the statistics of the colony concentrations of S. aureus (upper) and MRSA (lower) in the bacterial solution of each group.

[0020] Figure 3 This is a statistical result diagram of the diameters of the inhibition zones produced by PAP protein and vancomycin (Van) against S. aureus (left) and MRSA (right) in Example 1, respectively.

[0021] Figure 4 1 is a graph showing the effect of PAP protein on the proliferation and anti-apoptosis of RAW264.7 cells in Example 2; wherein A is a graph showing the statistical results of the effect of different concentrations of PAP protein on the proliferation of RAW264.7 cells in 24 hours, and B is a graph showing the statistical results of the effect of different concentrations of PAP protein on the anti-apoptosis of RAW264.7 cells.

[0022] Figure 5 2 is a graph showing the depolarization effect of PAP protein on RAW264.7 cells in Example 2; wherein A is an observation and recording graph of the cell morphology of each group under bright field conditions, B is a statistical graph showing the content of M1-type RAW264.7 cells, and C is a statistical graph showing the content of M2-type RAW264.7 cells.

[0023] Figure 6 This is a graph showing the detection results of the concentrations of inflammatory factors (TNF-α, IFN-γ, IL-6) and anti-inflammatory factors (IL-4) in RAW264.7 macrophages after LPS-induced polarization in Example 2.

[0024] Figure 7 The CFU and OD in the MRSA bacterial culture process in Example 3 600 relationship diagram.

[0025] Figure 8 3 is a graph showing the therapeutic effect of PAP protein on suppurative arthritis in BALB / c mice in Example 3; wherein A is a visual graph of the joints of each group at Day 0, 1, 4, and 8 of treatment, respectively; B is a statistical graph of daily body weight of each group of mice during the treatment period; and C is a statistical graph of daily joint size measurements of each group of mice during the treatment period.

[0026] Fig. 9 The figure is a diagram of the treatment effect evaluation of each group in Example 3 on Day 8 by the plantar inkblot method. DETAILED DESCRIPTION

[0027] The present invention is further described in detail below in conjunction with specific embodiments and accompanying drawings, but the protection content of the present invention is not limited to the following embodiments. Without departing from the spirit and scope of the present invention, changes and advantages that can be thought of by those skilled in the art are included in the present invention, and the attached claims are the scope of protection. The process, conditions, reagents, experimental methods, etc. for implementing the present invention, except for the contents specifically mentioned below, are all common knowledge and common common sense in the art, and the present invention does not particularly limit the content.

[0028] The PAP protein used in the following examples is a recombinant PAP protein, which is recombinantly prepared with reference to the existing literature [Yu LT, Yang MQ, Liu JL, et al. Recombinant Reg3α protein protects against experimental acute pancreatitis in mice. Mol Cell Endocrinol. 2016 Feb 15; 422: 150-159. doi: 10.1016 / j.mce.2015.12.002. Epub 2015 Dec 9. PMID: 26683606.].

[0029] Example 1: In vitro antibacterial effect of PAP protein

[0030] (1) Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC):

[0031] In a 96-well plate, the PAP protein solution was diluted to 1024, 512, 256, 128, 64, 32, 16, and 8 μg / mL using LB liquid medium, and a blank control group and a bacterial solution control group were set up, with 3 replicates in each group. Each well was inoculated with 4×10 6 CFU of S. aureus and MRSA were placed in a 37°C incubator and incubated for 18-24 hours, and then the OD was measured. 600 Absorbance value.

[0032] from Figure 1 As can be seen from the plate intuitive diagram of Figure A, for S. aureus and MRSA, when the PAP protein concentration is ≤32μg / mL and 64μg / mL respectively, obvious turbidity can be seen with the naked eye. Figure 1 In Figure C, the absorbance values ​​of each well at 600 nm show that for S. aureus and MRSA, the groups of PAP protein ≤32 μg / mL and 64 μg / mL are significantly different from the blank (sterile) group, indicating that the MIC values ​​of PAP protein for S. aureus and MRSA are 64 μg / mL and 128 μg / mL, respectively. By coating the solution in each well, it can be seen that for S. aureus ( Figure 1 Colonies were formed only when the PAP protein concentration was lower than 64 μg / mL; MRSA ( Figure 1 (Figure 1B, upper panel) Colonies were formed only when the PAP protein concentration was lower than 128 μg / mL, which was consistent with the above results and further verified that the MIC values ​​of PAP protein for S. aureus and MRSA were 64 μg / mL and 128 μg / mL, respectively. ** p<0.01, ***p<0.001, One-way ANOVA).

[0033] Take 100 μL of solution from each well of the 96-well plate and apply it on the plate or dilute it to the corresponding multiple and then apply it on the plate, and incubate it in a 37°C incubator for 24 hours. Finally, use ImageJ software to count the number of colonies.

[0034] like Figure 2 As shown, the statistical graph of the number of colonies diluted to the effective counting range shows that for S. aureus and MRSA, the PAP protein groups ≤32μg / mL and 64μg / mL, respectively, are significantly different from the blank (sterile) group, indicating that the MBC values ​​of PAP protein for S. aureus and MRSA are 64μg / mL and 128μg / mL, respectively. ( *** p<0.001, One-way ANOVA).

[0035] (2) Determination of inhibition zone by disc diffusion method

[0036] S. aureus and MRSA activated overnight were inoculated into test tubes containing LB liquid culture medium, and cultured at 37°C and 220rpm for 3 hours. The cultured bacterial solution was diluted to an appropriate concentration and evenly coated on the LB solid plate. A negative control (PBS) group, a group of PAP protein administration with different concentrations, and a positive control (vancomycin) group were set up, and 15 μL of each solution was dripped onto a sterile blank paper disk in the LB solid plate and dried until there was no obvious solution. Then it was placed in a 37°C incubator for incubation for 12-24 hours, and the diameter of each inhibition zone was measured using a digital vernier caliper. Each group of experiments was repeated three times.

[0037] like Figure 3 As shown, the PAP protein in the high and medium dose groups had good inhibitory effects on both S. aureus and MRSA, and compared with vancomycin, the inhibitory effect of PAP protein in the high and medium dose groups on S. aureus was better than that of vancomycin; the inhibitory effect of PAP protein in the high dose group on MRSA was equivalent to that of vancomycin. ( *** p<0.001, One-way ANOVA).

[0038] The above results indicate that PAP protein has a good inhibitory effect on S. aureus and MRSA in vitro.

[0039] Example 2: Immunomodulatory effect of PAP protein on mouse-derived RAW264.7 macrophages in vitro

[0040] (1) Proliferation-promoting and anti-apoptotic effects of PAP protein on RAW264.7 cells

[0041] RAW264.7 cells were cultured in a 37°C 5% CO2 incubator with DMEM containing 10% heat-inactivated fetal bovine serum and 1% penicillin-streptomycin. When the cell density reached 75%-80%, the cells were incubated for 10 min. 4 Cells were seeded in 96-well plates at a density of 10 cells / well. The cells were incubated overnight, and then different concentrations of PAP protein were added for 24 hours. Cell viability was evaluated using the MTT assay, and the absorbance at 570 nm was measured by a microplate reader. 4 The cells were inoculated at a density of 10 cells / well in a 96-well plate and cultured overnight. When the cell density was appropriate, different concentrations of LPS were added to induce cell apoptosis (three replicates were set for each group), and the MTT method was used to determine the optimal concentration of LPS to induce cell apoptosis. Then, a pre-protection experiment was performed at this concentration, that is, the cells were first co-cultured with different concentrations of PAP protein, and then the optimal concentration of LPS was added to induce cell apoptosis, and finally the cell survival rate was determined by the MTT method.

[0042] like Figure 4 As shown, PAP protein at a concentration of 10 nM showed a good effect of promoting RAW264.7 cell proliferation at 24 hours: Under the optimal LPS-induced RAW264.7 cell apoptosis condition, PAP protein at a concentration of 1 nM had the best anti-RAW264.7 cell apoptosis effect. ( * p<0.05, ** p<0.01, One-way ANOVA).

[0043] (2) PAP protein depolarizes RAW264.7 cells to M1

[0044] 2×10 6 The cells were inoculated at a density of 10 cells / well in a 6-well plate and cultured overnight. When the cell density reached an appropriate level, LPS was used to induce the cells to polarize toward M1 type and a blank control was set. Then, different concentrations of PAP protein were added and cultured for 24 hours. Bright field images were collected under a microscope, and the cells of different polarities were counted and analyzed using ImageJ software.

[0045] like Figure 5 As shown, under bright field conditions, LPS induced cells to transform into M1 type, and the morphology changed significantly, becoming flat and octopus-shaped. After administration, the number of M1 macrophages in the high, medium and low dose groups decreased. As shown in the statistical results, there were significant differences between the different concentrations of administration groups and the induced groups. The results showed that PAP protein had a depolarizing effect on RAW264.7 cells. ( * p<0.05, ** p<0.01, *** p<0.001, One-way ANOVA).

[0046] (3) Detection of inflammatory factors

[0047] Take 10 4 The cells were inoculated at a density of 10 cells / well in a 96-well plate and cultured overnight. They were induced with 200 ng / mL LPS for 24 hours, the supernatant was removed, and different concentrations of PAP protein solutions prepared with incomplete culture medium were added. The cells were cultured for 24 hours, the supernatant was centrifuged, and the concentrations of inflammatory factors in each group were detected using enzyme-linked immunosorbent assay kits for TNF-α, IFN-γ, IL-6 and IL-4. Three replicate wells were set for each group.

[0048] like Figure 6 As shown in the figure, after LPS-induced polarization of RAW264.7 macrophages, after being treated with different concentrations of PAP protein, it was found that compared with the non-administered group, the concentration of inflammatory factors (TNF-α, IFN-γ, IL-6) in the administered group was reduced; the concentration of anti-inflammatory factors (IL-4) was increased, and the most significant anti-inflammatory effect was achieved when the PAP protein administration concentration was 10nM, indicating that PAP protein treatment can increase the concentration of anti-inflammatory factors and restore inflammatory factors to normal levels. ( * p<0.05, ** p<0.01, *** p<0.001, One-way ANOVA).

[0049] The above results indicate that PAP protein has an immunomodulatory effect on mouse-derived RAW264.7 macrophages.

[0050] Example 3: Therapeutic effect of PAP protein on suppurative arthritis in mice

[0051] (1) Bacterial culture

[0052] MRSA was streaked on solid LB medium. After colonies grew, a single colony was picked and inoculated into liquid LB medium. The culture was shaken at 37°C and 220 rpm overnight. The bacteria activated by overnight culture were inoculated into fresh medium at 1%, and cultured at 37°C and 220 rpm. The OD600 value of bacterial growth was detected at 1, 1.5, 2, 2.5, 3, and 3.5 hours, and the bacterial CFU and OD were plotted. 600 The relationship equation of the value.

[0053] like Figure 7 As shown, bacterial CFU and OD 600 The relationship is: Y = 0.0094x-0.1859, R 2 =0.8637. From the relationship, we can roughly calculate that after 3 hours of culture, the bacterial solution is diluted three times with physiological saline, and 10 μL is taken, which means 4×10 6 The CFU of bacteria is convenient for the subsequent calculation of the bacterial dose for mouse modeling.

[0054] (2) Establishment of septic arthritis in BALB / c mice

[0055] Four groups were set up, namely, blank (normal saline) group, MRSA model group, MRSA model + vancomycin (Van) treatment group, and MRSA model + PAP protein treatment group. Except for the blank group, 4×10 6 CFU of MRSA, one day later, obvious edema appeared in the joints, which means the model was successful. Figure 8 As shown. ( * p<0.05, One-way ANOVA).

[0056] (3) The therapeutic effect of PAP protein on suppurative arthritis in BALB / c mice

[0057] The PAP treatment group was injected with 180 μg / kg of PAP protein solution every day in the right leg of the model according to the weight of the mice. The positive control group was injected with 200 μg / kg of vancomycin every day in the right leg of the model according to the weight of the mice. The blank group and the MRSA modeling group were injected with the same volume of saline. The weight and joint swelling of the mice were monitored and counted every day. In addition, on the eighth day of treatment, the four groups of mice were subjected to the plantar inkblot experiment to analyze the gait of the mice. The specific operation was to apply ink on the soles of the mice, place them on a 15 cm white paper, and let them crawl. A one-week simulation experiment was carried out before the formal experiment to ensure that the mice passed smoothly without external stimulation and to ensure the authenticity and feasibility of the data.

[0058] like Figure 8 As shown in the figure, the joints of mice were significantly swollen after modeling, and the joint swelling persisted in the modeling group without treatment. As the treatment progressed, both the PAP protein group and the Van group were able to effectively improve joint edema. On Day 10, the joints of mice in the PAP protein group were completely swollen before those in the Van group. On the last day of treatment, the modeling group was still swollen, which was significantly different from the PAP protein group with reduced swelling. The above results show that the PAP protein at this concentration has a better therapeutic effect than Van. ( * p<0.05, One-way ANOVA).

[0059] Fig. 9 The plantar inkblot method also proved this point. The inkblots on the right paws of the PAP protein group were obvious and complete, comparable to the blank control group, while the inkblots on the right paws of the Van treatment group were incomplete, indicating that the mice may still be in pain and unwilling to put their feet. The MRSA model group mice were the most serious, with unclear inkblots, messy footsteps, and even tail dragging.

[0060] In summary, PAP protein has a good therapeutic effect on suppurative arthritis in mice.

Claims

1. Use of pancreatitis-related proteins or their derivatives in the preparation of drugs for the treatment of suppurative arthritis.

2. The use according to claim 1, characterized in that: The suppurative arthritis is suppurative arthritis caused by infection with Staphylococcus aureus and / or methicillin-resistant Staphylococcus aureus.

3. The use according to claim 1, characterized in that: The pancreatitis-associated protein is a natural pancreatitis-associated protein or a recombinant pancreatitis-associated protein constructed by genetic engineering.

4. The use according to claim 3, characterized in that: Natural pancreatitis-related proteins are pancreatitis-related proteins extracted from animal tissues or digestive fluids, or natural pancreatitis-related proteins fermented by microorganisms; recombinant pancreatitis-related proteins are recombinant pancreatitis-related proteins fermented by microorganisms.

5. The use according to claim 4, characterized in that: The animal is mouse, rat, guinea pig, cow, pig, dog or rabbit, the animal tissue is pancreatic tissue or intestinal tissue, and the digestive juice is animal pancreatic juice, intestinal juice or bile.

6. The use according to claim 1, characterized in that: The dosage form of the drug for treating suppurative arthritis is an injectable dosage form; the administration method of the drug for treating suppurative arthritis is local injection or systemic injection.

7. The use according to claim 6, characterized in that: Local injections are given subcutaneously or intra-articularly.

8. The use according to claim 1, characterized in that: The subject of administration of the drug for treating suppurative arthritis is a human or non-human animal who may develop or has developed suppurative arthritis.

9. The use according to claim 1, characterized in that: The dose of the drug used to treat septic arthritis is 100~1000 μg / kg.

10. The use according to claim 1, characterized in that The dose of the drug administered for the treatment of septic arthritis is 180 μg / kg.

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