Use of cpi2 protein in the preparation of a medicament for the treatment of gouty arthritis

Abstract

The application discloses application of CPI2 protein in preparation of anti-gouty arthritis drugs and belongs to the technical field of biological medicine. The application discloses the application of the CPI2 protein in preparation of anti-gouty arthritis drugs, and the amino acid sequence of the CPI2 protein is shown as SEQ ID NO. 1. The CPI2 protein disclosed by the application can reduce joint swelling caused by sodium urate, reduce inflammatory cell infiltration, reduce the levels of proinflammatory cytokines IL-1beta, IL-6 and TNF-alpha, increase the level of anti-inflammatory cytokine IL-10, increase the activities of SOD and GSH-Px, and reduce the content of MDA, and can be prepared as a drug for preventing or treating gouty arthritis and has clinical application value.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically to the application of CPI2 protein in the preparation of drugs for treating gouty arthritis. Background Technology

[0002] Gout, caused by the deposition of urate crystals in joints and surrounding tissues, is one of the most common inflammatory arthritis worldwide. Patients experience elevated blood uric acid levels, and contributing factors include a high-purine diet, alcohol consumption, obesity, and abnormal renal excretion of uric acid. In modern clinical practice, gout treatment primarily relies on nonsteroidal anti-inflammatory drugs (NSAIDs), colchicine, and glucocorticoids. However, it is important to note that long-term or excessive use of these drugs often leads to a series of serious adverse reactions, such as gastrointestinal toxicity, nephrotoxicity, and gastrointestinal bleeding, and their therapeutic effects have inherent limitations. These adverse reactions not only impose an additional burden on patients' health but also significantly reduce their acceptance of traditional treatments. Given the numerous problems with current gout medications, there is an urgent need to explore the anti-inflammatory mechanisms of gout and to find new, safe, and effective treatments to provide better treatment options for gout patients.

[0003] Parasites, living within their hosts, have developed molecules that regulate the host's immune system to evade immune attacks. These molecules may be developed into novel anti-inflammatory drugs. Cysteine ​​protease inhibitors (CPIs) are believed to play a significant role in parasites evading the host's immune response and may be used to prevent and treat diseases such as inflammatory bowel disease (IBD), allergies, and rheumatoid arthritis (RA). Rheumatoid arthritis is caused by autoimmune abnormalities (the immune system attacking the synovial membrane of joints). Patients typically have normal blood uric acid levels. Contributing factors include genetics, infection, smoking, and abnormal hormone levels. Treatment primarily involves methotrexate, leflunomide, and hydroxychloroquine, which need to be used early to slow joint destruction. Biologics may be used for severe cases or those unresponsive to conventional treatments. Clearly, gouty arthritis and rheumatoid arthritis differ significantly in their pathogenesis, contributing factors, and treatments.

[0004] The research team of this invention has long been dedicated to studying bioactive molecules in parasites. A series of protein / peptide bioactive components have been screened from the human intestinal nematode, hookworm of the duodenum, including four cysteine ​​protease inhibitors. Among them, cysteine ​​protease inhibitor 2 (CPI2) exhibits significant inhibitory activity and good selectivity against cathepsin S (CTSS). Cathepsin S (CTSS) is a unique lysosomal protease that occupies a special position in the cysteine ​​cathepsin family and is involved in the pathological processes of various diseases, including cancer, cardiovascular disease, and arthritis.

[0005] Therefore, providing the application of CPI2 protein in the preparation of drugs for treating gouty arthritis is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides the application of CPI2 protein in the preparation of drugs for treating gouty arthritis.

[0007] This invention found that CPI2 has a significant protective effect against sodium urate-induced gouty arthritis in mice, indicating that CPI2 can be used clinically as an anti-gouty arthritis drug.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] The application of CPI2 protein in the preparation of drugs for treating gouty arthritis, wherein the amino acid sequence of CPI2 protein is shown in SEQ ID NO.1.

[0010] Furthermore, a pharmaceutical formulation for treating gouty arthritis comprises CPI2 protein as an active ingredient, either directly or with a pharmaceutically acceptable carrier; the amino acid sequence of said CPI2 protein is shown in SEQ ID NO.1.

[0011] CPI2 amino acid sequence:

[0012] SEQ ID NO.1.

[0013] As can be seen from the above technical solution, compared with the prior art, this invention discloses the application of CPI2 protein in the preparation of anti-gouty arthritis drugs. Using a mouse model of gouty arthritis induced by sodium urate, CPI2 significantly improved the degree of swelling in the model mice, reduced the infiltration of inflammatory cells in the serum, decreased the levels of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α, increased the level of anti-inflammatory cytokine IL-10, increased the activity of SOD and GSH-Px, and decreased the content of MDA. CPI2 can significantly improve the symptoms of sodium urate-induced gouty arthritis, reduce inflammatory response and oxidative stress damage, and its improvement effect is even better than that of colchicine, a commonly used clinical drug, indicating that CPI2 can be used clinically as an anti-gouty arthritis drug. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0015] Figure 1 The effect of CPI2 on the thickness of the right footpad in a mouse model of sodium urate-induced gouty arthritis ( (n=10);

[0016] Wherein, A is the blank control group; B is the model group; C is the colchicine group; D is the low-dose CPI2 group; E is the medium-dose CPI2 group; F is the high-dose CPI2 group; G is the thickness level of the right paw pad of mice in each group; ** P<0.01, compared with the blank control group; # P<0.05, ## P<0.01, compared with the model group; @P<0.05, compared with the medium-dose CPI2 group; ^^ P<0.01, compared with the high-dose CPI2 group;

[0017] Figure 2 The effect of CPI2 on the pathological morphology of the right footpad in a mouse model of sodium urate-induced gouty arthritis ( (n=10);

[0018] Figure 3 The effect of CPI2 on serum inflammatory factor levels in a mouse model of sodium urate-induced gouty arthritis ( (n=10);

[0019] Where A represents the serum TNF-α level of each group of mice; B represents the serum IL-1β level of each group of mice; C represents the serum IL-6 level of each group of mice; and D represents the serum IL-10 level of each group of mice. ** P<0.01, compared with the blank control group; # P<0.05, ## P<0.01, compared with the model group; && P<0.01, compared with the low-dose CPI2 group; @P<0.05, @@P<0.01, compared with the medium-dose CPI2 group;

[0020] Figure 4 The effect of CPI2 on serum oxidative stress injury markers in a mouse model of sodium urate-induced gouty arthritis ( (n=10);

[0021] Where A represents the serum GSH-Px level of each group of mice; B represents the serum SOD level of each group of mice; and C represents the serum MDA level of each group of mice. ** P<0.01, compared with the blank control group; ## P<0.01, compared with the model group; && P<0.01, compared with the low-dose CPI2 group; @@P<0.01, compared with the medium-dose CPI2 group. Detailed Implementation

[0022] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] Example 1: Study on the effect of CPI2 in improving sodium urate-induced gouty arthritis in mice

[0024] 1) Animals

[0025] Sixty two-month-old male C57BL / 6 mice were purchased from Liaoning Changsheng Biotechnology Co., Ltd., and were used for experiments after one week of acclimatization. Animal qualification certificate number: SCXK(Liaoning)2020-0001. The experimental protocol was approved by the Animal Experiment Ethics Committee of Guangdong Medical University, ethics approval number: GDMU-2023-000061.

[0026] 2) Instruments

[0027] The Cytation 5 multi-functional microplate reader was purchased from OmegaBio Tek, USA; the KD2268 microtome was purchased from Kedi Instrument Co., Ltd., Jinhua City, Zhejiang Province.

[0028] 3) Drugs and reagents

[0029] Colchicine was purchased from Hongyun Pharmaceutical Group Co., Ltd., batch number: 230106; uric acid was purchased from Sigma-Aldrich (USA), batch number: BCCH2974; ELISA kits for tumor necrosis factor alpha (TNF-α), interleukin 1β (IL-1β), interleukin 6 (IL-6), and interleukin 10 (IL-10) were purchased from R&D Company (USA), batch numbers P340968, P138448, P311746, and P322031, respectively; glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) were also tested. The detection kits for dismutase (SOD) and malondialdehyde (MDA) were purchased from Nanjing Jiancheng Biotechnology Institute, with batch numbers 20211111, 20201127 and 20180315, respectively.

[0030] Recombinant preparation of CPI2: The previously constructed E. coli strain of CPI2 recombinant protein [Reference: Shao Zheng, et al. Isolation, expression and activity study of hookworm cysteine ​​protease inhibitor. Chinese Journal of Pathogenic Biology, 2022, 17(11):1278-1282] was inoculated into LB liquid medium containing ampicillin (100 μg / mL) and cultured at 37℃ and 150 rpm until the optical density (OD) was approximately 0.6. Subsequently, isopropyl β-D-1-thiogalactopyranoside (IPTG, 100 μg / mL) was added, and expression was induced at 35℃ for 6 h. After induction, the bacterial cells were collected by centrifugation at 6000 rpm for 30 min. The bacterial cells were resuspended in LEW buffer (17.9 g Na₂HPO₄·12H₂O and 17.5 g NaCl were weighed, dissolved in 800 mL of double-distilled water, the pH was adjusted to 7.0 with sodium hydroxide, and then double-distilled water was added to bring the volume to 1 L), and then sonicated. Finally, the mixture was centrifuged at 12000 rpm for 30 min, and the supernatant was collected. The supernatant was thoroughly mixed with Ni-NTA resin at a ratio of 20:1 (v / v), and then washed with 10–20 column volumes of washing buffer (50 mM PBS, 0.3 M NaCl, 30 mM imidazole) to remove unbound impurities. The fusion protein was digested on-column with SUMO protease at a molar ratio of 150:1, and finally eluted with 5–10 column volumes of LEW buffer. CPI2 obtained by Ni-NTA affinity chromatography was further purified using SP Bio-sep FF ion exchange chromatography to obtain endotoxin-free recombinant protein CPI2.

[0031] CPI2 amino acid sequence:

[0032] SEQ ID NO.1.

[0033] 4) Preparation of sodium urate crystals

[0034] Add 25g of uric acid to 200mL of boiling water, and simultaneously add 6.0mL of 1mol / L NaOH solution to dissolve it. Then, carefully adjust the pH of the resulting solution to 7.2 with hydrochloric acid. Place the solution in a refrigerator at 4°C overnight to allow crystals to precipitate. The next day, separate the precipitate from the solution by filtration, and dry the precipitate at 37°C.

[0035] 5) Mouse model of gouty arthritis induced by sodium urate

[0036] Mice were anesthetized by inhalation of 5% isoflurane. Subsequently, sodium urate (2 mg dissolved in 40 μL of physiological saline) was injected into the right paw pad of each mouse. Mice in the sham-operated group received an equal volume of sterile physiological saline injected into their right paw pad.

[0037] 6) Grouping and Dosing

[0038] All mice were randomly divided into 6 groups of 10 mice each: a blank control group, a model group, a colchicine group (0.5 mg / kg), a low-dose CPI2 group (0.5 mg / kg), a medium-dose CPI2 group (1 mg / kg), and a high-dose CPI2 group (2 mg / kg). Except for the blank control group, which received an injection of 40 μL of saline in its right paw pad, the other groups were induced to develop a gouty arthritis mouse model by injecting 40 μL of sodium urate solution (2 mg dissolved in 40 μL of saline) into their right paw pads. After modeling, the colchicine group was administered colchicine via gavage, while the CPI2 group received CPI2 via tail vein injection. Samples were collected 24 hours after modeling.

[0039] 7) Draw materials

[0040] Twenty-four hours after modeling, the thickness of the right paw pad of each group of mice was measured using electronic calipers. Mice were anesthetized by inhalation of 5% isoflurane, and blood was collected by enucleation. The blood was left to stand at room temperature for 1 hour, centrifuged at 3000 r / min for 15 min, and the serum was separated and stored at -20℃ for later testing. The right paw pad tissue was separated and preserved by soaking in 10% neutral formaldehyde.

[0041] 8) Morphological observation of the right footpad

[0042] Sagittal sections were prepared from the right footpad after fixation in 10% neutral formaldehyde for 48 h. The sections were decalcified in 0.5 MEDTA-PBS solution (pH 7.8) at room temperature for 14 days, rinsed overnight with running water, dehydrated in a gradient of 70%-100% ethanol, cleared in xylene for 5 min, embedded in paraffin, sectioned to a thickness of 6 μm, and then stained with hematoxylin and eosin. The sections were then observed and photographed under a microscope.

[0043] 9) Detection of serum inflammatory factors and oxidative stress-related indicators

[0044] Mouse serum was collected, and the levels of TNF-α, IL-1β, IL-6, IL-10, MDA, and the activities of SOD and GSH-Px in the serum of each group of mice were detected according to the detection steps in the kit instructions.

[0045] 10) Statistical methods

[0046] All measurement data are expressed as Statistical analysis was performed using Prism v.8.0 software. One-way ANOVA was used for comparisons among multiple groups. A p-value < 0.05 was considered statistically significant.

[0047] 11) Results

[0048] Figure 1 The effect of CPI2 on the thickness of the right footpad in a mouse model of sodium urate-induced gouty arthritis; Figure 1 The results showed that, compared with the blank control group, the thickness of the right paw pad of mice in the model group was significantly increased (P < 0.01). Compared with the model group, the thickness of the right paw pad of mice in the colchicine group and the CPI2 administration group was significantly decreased (P < 0.05). Compared with the medium-dose CPI2 group, the thickness of the right paw pad of mice in the colchicine group was significantly increased (P < 0.05). Compared with the high-dose CPI2 group, the thickness of the right paw pad of mice in the colchicine group was significantly increased (P < 0.01).

[0049] Figure 2 The effect of CPI2 on the pathological morphology of the right footpad in a mouse model of sodium urate-induced gouty arthritis; Figure 2 The results showed that, compared with the blank control group, the inflammatory cell infiltration in the right paw pad of mice in the model group was significantly increased. Compared with the model group, the inflammatory cell infiltration in the right paw pad of mice in the colchicine group and the CPI2 administration group was significantly reduced. Among them, the inhibition of inflammatory cell infiltration in the CPI2 administration group was dose-dependent.

[0050] Figure 3 The effect of CPI2 on serum inflammatory factor levels in a mouse model of sodium urate-induced gouty arthritis; Figure 3The results showed that, compared with the blank control group, the serum levels of TNF-α, IL-1β, IL-6, and IL-10 in the model group mice were significantly increased (P < 0.01). Compared with the model group, the serum levels of TNF-α, IL-1β, and IL-6 in the colchicine group and the CPI2 administration group were significantly decreased (P < 0.01), while the IL-10 level was significantly increased (P < 0.05). Compared with the low-dose CPI2 group, the serum levels of TNF-α, IL-1β, and IL-6 in the colchicine group, the medium-dose CPI2 group, and the high-dose CPI2 group were significantly decreased (P < 0.01), while the serum IL-10 level in the high-dose CPI2 group was significantly increased (P < 0.01). Compared with the medium-dose CPI2 group, the serum levels of TNF-α, IL-1β, and IL-6 in the high-dose CPI2 group were significantly decreased (P < 0.05), while the IL-10 level was significantly increased (P < 0.01).

[0051] Figure 4 The effect of CPI2 on serum oxidative stress injury indicators in a mouse model of sodium urate-induced gouty arthritis; Figure 4 The results showed that, compared with the blank control group, the serum GSH-Px and SOD activities of mice in the model group were significantly decreased (P < 0.01), while the MDA level was significantly increased (P < 0.01). Compared with the model group, the serum GSH-Px and SOD activities of mice in the colchicine group and the CPI2 administration group were significantly increased (P < 0.01), while the MDA level was significantly decreased (P < 0.01). Compared with the low-dose CPI2 group, the serum GSH-Px and SOD activities of mice in the colchicine group, the medium-dose CPI2 group, and the high-dose CPI2 group were significantly increased (P < 0.01), while the MDA level was significantly decreased (P < 0.01). Compared with the medium-dose CPI2 group, the serum GSH-Px and SOD activities of mice in the high-dose CPI2 group were significantly increased (P < 0.01).

[0052] 12) Conclusion

[0053] CPI2 can significantly improve the symptoms of gouty arthritis induced by sodium urate and reduce sodium urate-induced inflammatory response and oxidative stress damage. Therefore, CPI2 can be used to prepare drugs for the prevention and treatment of gouty arthritis.

[0054] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The application of CPI2 protein in the preparation of drugs for treating gouty arthritis, characterized in that, The amino acid sequence of the CPI2 protein is shown in SEQ ID NO.1.

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