Application of CPI2 protein in preparation of anti-gouty arthritis drugs

Abstract

The invention discloses application of CPI2 protein in preparation of anti-gouty arthritis drugs, and belongs to the technical field of biological medicines. According to the application of the CPI2 protein in preparation of the gouty arthritis resisting medicine, the amino acid sequence of the CPI2 protein is shown as SEQ ID NO.1. The invention further discloses an application of the CPI2 protein in preparation of the gouty arthritis resisting medicine. The CPI2 protein disclosed by the invention can relieve 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 cytokines IL-10, increase the activity of SOD and GSH-Px and reduce the content of MDA, can be used for preparing medicines for preventing or treating gouty arthritis, and has clinical application value.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and more specifically to the application of CPI2 protein in the preparation of anti-gouty arthritis drugs. Background Art

[0002] Gout is a disease caused by the deposition of urate salts in joints and surrounding tissues, leading to lesions and inflammatory reactions, and is one of the most common inflammatory arthritides globally. The serum uric acid level of patients increases, and the inducing factors include high-purine diet, alcohol consumption, obesity, and abnormal renal excretion function of uric acid. In modern clinical practice, the treatment of gout mainly relies on drugs such as non-steroidal anti-inflammatory drugs, colchicine, and glucocorticoids. However, it should be noted that long-term or excessive use of these drugs often causes 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 bring an additional burden to the health of patients but also significantly reduce the acceptance of traditional therapeutic drugs by patients. Given the many problems existing in current gout treatment drugs, it is urgent to deeply explore the anti-inflammatory mechanism of gout, and it is necessary to find new safe and effective therapeutic drugs to provide better treatment options for gout patients.

[0003] Parasites parasitize in the host body and have developed molecules to regulate the host immune system to avoid immune killing by the host, and these molecules may be developed into new anti-inflammatory drugs. Parasite cysteine protease inhibitors (CPI) are considered to play an important role in the parasite's escape from the host immune response and may be used to prevent and treat diseases such as inflammatory bowel disease (IBD), allergy, and rheumatoid arthritis (RA). Rheumatoid arthritis is caused by abnormal autoimmunity (the immune system attacks the synovium of joints), and the serum uric acid level of patients is normal. The inducing factors include genetics, infection, smoking, abnormal hormone levels, etc. The main treatment drugs are methotrexate, leflunomide, hydroxychloroquine, etc., and they need to be used early to delay joint destruction. For patients with severe conditions or ineffective conventional treatments, biological agents can be used. Obviously, gouty arthritis and rheumatoid arthritis are completely different in terms of pathogenesis, inducing factors, and treatment drugs.

[0004] The research team of the present invention has been long-term committed to the research of bioactive molecules in parasites. A series of protein / polypeptide active components have been screened from the human intestinal nematode - Ancylostoma duodenale, including 4 cysteine protease inhibitors, among which cysteine protease inhibitor 2 (CPI2) has a significant inhibitory effect on cathepsin S (cat S, or CTSS) and shows good selectivity. Cathepsin S (CTSS) is a unique lysosomal protease and occupies a special position in the cysteine cathepsin family. It is involved in the pathological processes of various diseases such as cancer, cardiovascular diseases, and arthritis.

[0005] Therefore, providing the application of CPI2 protein in the preparation of anti-gouty arthritis drugs is an urgent problem 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 anti-gouty arthritis drugs.

[0007] The present invention discovers that CPI2 has a significant protective effect on sodium urate-induced gouty arthritis in mice, indicating that CPI2 can be used as an anti-gouty arthritis drug in clinical practice.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

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

[0010] Furthermore, a pharmaceutical preparation for anti-gouty arthritis, which uses CPI2 protein as an active ingredient and formulates it directly or with a pharmaceutically acceptable carrier; the amino acid sequence of the CPI2 protein is as shown in SEQ ID NO.1.

[0011] CPI2 amino acid sequence:

[0012] QVMTGGVMTQDPSDPEYMKKAWKAAITLNQESNVKYLMVPIKVVK ADSQVVGGMKYTFEVLFGQSECNKGDVELSKLATANCQLIPNGSRALYK VELYERLWENFEQYTVTKIKDVSA; SEQ ID NO.1.

[0013] According to the above technical solutions, compared with the prior art, the present invention discloses the application of CPI2 protein in the preparation of anti-gouty arthritis drugs. By using a sodium urate-induced gouty arthritis mouse model, CPI2 can significantly improve the swelling degree of model mice, reduce the infiltration of inflammatory cells in serum, lower the levels of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α, increase the level of anti-inflammatory cytokine IL-10, increase the activities of SOD and GSH-Px, and reduce the content of MDA. CPI2 can significantly improve the symptoms of sodium urate-induced gouty arthritis, reduce the inflammatory response and oxidative stress injury, and its improvement effect is better than that of colchicine, a commonly used drug in clinical practice at present, indicating that CPI2 can be used as an anti-gouty arthritis drug in clinical practice. Brief Description of the Drawings

[0014] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the accompanying drawings required in the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can also be obtained according to the provided drawings.

[0015] Figure 1 Effect of CPI2 on the right hind paw pad thickness in a mouse model of sodium urate-induced gouty arthritis ( n = 10);

[0016] Among them, 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 right hind paw pad thickness 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 < 0.05, compared with the medium-dose CPI2 group; ^^ P < 0.01, compared with the high-dose CPI2 group;

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

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

[0019] Among them, A is the serum TNF-α level of each group of mice; B is the serum IL-1β level of each group of mice; C is the serum IL-6 level of each group of mice; D is 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 Effect of CPI2 on the levels of serum oxidative stress injury indexes in a mouse model of sodium urate-induced gouty arthritis ( n = 10);

[0021] Among them, A is the serum GSH-Px level of mice in each group; B is the serum SOD level of mice in each group; C is the serum MDA level of mice in each group; ** 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. Specific implementation manners

[0022] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

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

[0024] 1) Animals

[0025] 60 male C57BL / 6 mice at 2 months old were purchased from Liaoning Changsheng Biotechnology Co., Ltd. After 1 week of adaptive feeding, the experiment was carried out. Animal certificate number: SCXK(Liao)2020-0001. The experimental protocol was approved by the Animal Experiment Ethics Committee of Guangdong Medical University, and the ethics approval number: GDMU-2023-000061.

[0026] 2) Instruments

[0027] The Cytation 5 type multifunctional microplate reader was purchased from omegaBio Tek Company, USA; the KD2268 type microtome was purchased from Cody Instruments 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 Company in the United States, batch number: BCCH2974; Tumor necrosis factor alpha (TNF-α), Interleukin 1beta (IL-1β), Interleukin 6 (IL-6) and Interleukin 10 (IL-10) ELISA kits were purchased from R&D Company in the United States, and the batch numbers were P340968, P138448, P311746 and P322031 respectively; Glutathione Peroxidase (GSH-Px), Superoxide dismutase (SOD) and Malondialdehyde (MDA) detection kits were purchased from Nanjing Jiancheng Bioengineering Institute, and the batch numbers were 20211111, 20201127 and 20180315 respectively.

[0030] Recombinant preparation of CPI2: The Escherichia coli (E. coli) strain of the previously constructed CPI2 recombinant protein [References: Shao Zheng, et al. Isolation, expression and activity study of cysteine protease inhibitor from Ancylostoma duodenale. Chinese Journal of Pathogen Biology, 2022, 17(11): 1278-1282] was inoculated into LB liquid medium containing ampicillin (100 μg / mL) and cultured at 37 °C 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 induced at 35 °C for 6 h. After the induction, the cells were collected by centrifugation at 6000 rpm for 30 min. The cell pellet was resuspended in LEW buffer (Weigh 17.9 g of Na2HPO4·12H2O and 17.5 g of NaCl, dissolve in 800 ml of double-distilled water, adjust the pH value to 7.0 with sodium hydroxide, and then make up the volume to 1 L with double-distilled water), and then sonicated. Finally, centrifuged at 12000 rpm for 30 min to collect the supernatant. The supernatant was mixed with Ni-NTA resin at a ratio of 20:1 (v / v), and then the unbound impurities were washed away with 10-20 column volumes of washing buffer (50 mM PBS, 0.3 M NaCl, 30 mM imidazole). The fusion protein was digested with SUMO protease on the column at a molar ratio of 150:1, and finally the target protein was eluted with 5-10 column volumes of LEW buffer. The CPI2 obtained by Ni-NTA affinity chromatography was further purified by SP Bio-sep FF ion exchange chromatography to obtain the endotoxin-free recombinant protein CPI2.

[0031] CPI2 amino acid sequence:

[0032] QVMTGGVMTQDPSDPEYMKKAWKAAITLNQESNVKYLMVPIKVVK ADSQVVGGMKYTFEVLFGQSECNKGDVELSKLATANCQLIPNGSRALYK VELYERLWENFEQYTVTKIKDVSA; SEQ ID NO.1.

[0033] 4) Preparation of sodium urate crystals

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

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

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

[0037] 6) Grouping and administration

[0038] All mice were randomly divided into 6 groups of 10 each, namely the blank control group, the model group, the colchicine group (0.5 mg / kg), the low-dose CPI2 group (0.5 mg / kg), the medium-dose CPI2 group (1 mg / kg), and the high-dose CPI2 group (2 mg / kg). Except for the mice in the blank control group, which were injected with 40 μL of physiological saline into the right hind paw, the mice in the other groups were injected with 40 μL of sodium urate solution (2 mg dissolved in 40 μL of physiological saline) into the right hind paw to induce a gouty arthritis mouse model. After modeling, the mice in the colchicine group were given the corresponding colchicine by gavage, and the CPI2 administration group was given CPI2 by tail vein injection. Samples were taken 24 h after modeling.

[0039] 7) Sample collection

[0040] Twenty-four hours after modeling, the thickness of the right hind paw of each group of mice was measured using an electronic caliper. Mice were anesthetized by inhaling 5% isoflurane, and blood was collected by orbital enucleation. The blood was allowed to stand at room temperature for 1 h, centrifuged at 3000 r / min for 15 min to separate the serum, which was stored at -20°C for further testing; the right hind paw tissue was separated and immersed in 10% neutral formaldehyde for preservation.

[0041] 8) Morphological observation of the right hind paw

[0042] The right hind paw pads fixed with 10% neutral formaldehyde for 48 h were sectioned sagittally. Decalcification was performed with 0.5 M EDTA-PBS solution (pH 7.8) at room temperature for 14 d, followed by rinsing with running water overnight, dehydration in a gradient of 70%-100% ethanol, clearing with xylene for 5 min, embedding in paraffin, sectioning with a thickness of 6 μm, and then hematoxylin-eosin staining of the sections, followed by microscopic observation and photography.

[0043] 9) Detection of serum inflammatory factors and levels of indicators related to oxidative stress injury

[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 mice in each group were detected according to the detection steps described in the kit instructions.

[0045] 10) Statistical methods

[0046] Measurement data are all expressed as Statistical analysis was performed using Prism v.8.0 software. One-way analysis of variance (ANOVA) was used for comparison among multiple groups. P < 0.05 was considered statistically significant.

[0047] 11) Results

[0048] Figure 1 Shows the effect of CPI2 on the thickness of the right hind paw pad 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 hind paw pad in the model group of mice increased significantly (P < 0.01). Compared with the model group, the thickness of the right hind paw pad in the colchicine group and the CPI2 administration group of mice decreased significantly (P < 0.05). Compared with the medium-dose CPI2 group, the thickness of the right hind paw pad in the colchicine group of mice increased significantly (P < 0.05). Compared with the high-dose CPI2 group, the thickness of the right hind paw pad in the colchicine group of mice increased significantly (P < 0.01).

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

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

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

[0052] 12) Conclusion

[0053] CPI2 can significantly improve the symptoms of sodium urate-induced gouty arthritis, and reduce the inflammatory reaction and oxidative stress injury induced by sodium urate. Therefore, CPI2 can be prepared for use in drugs for preventing and treating gouty arthritis.

[0054] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not 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. Use of CPI2 protein in the preparation of anti-gouty arthritis drugs, characterized in that, The amino acid sequence of the CPI2 protein is shown in SEQ ID NO.

1.

2. A pharmaceutical preparation for treating gouty arthritis, characterized in that, The CPI2 protein is formulated directly as an active ingredient or with a pharmaceutically acceptable carrier; the amino acid sequence of the CPI2 protein is shown in SEQ ID NO.1.

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