Anti-inflammatory polypeptide and application thereof

Abstract

The invention provides a mast cell threshing peptide with an anti-inflammatory effect and a functional analogue polypeptide thereof, or derivatives of the mast cell threshing peptide and the functional analogue polypeptide. Furthermore, the invention provides application of the polypeptide or the derivative thereof in preparation of drugs for preventing / treating inflammatory diseases.

Description

Technical Field

[0001] The present application belongs to the field of biomedicine, and specifically relates to the use of mast cell degranulation peptides and functional analog polypeptides thereof, or their derivatives in the preparation of agents with anti-inflammatory effects. Background Art

[0002] Bee sting therapy refers to a treatment method that uses the sting of bees to produce therapeutic effects. Bee sting therapy has a long history, with the Yellow Emperor's Classic of Internal Medicine, compiled during the Warring States Period, noting that "bee stings are toxic and can cure diseases." Historically, bee sting therapy has been widely used in Asia and Europe to treat ailments such as rheumatism, rheumatoid arthritis, osteoarthritis, lumbar and cervical spondylosis, and ankylosing spondylitis.

[0003] Systematic research on bee sting therapy began in the late 19th century. In 1888, Viennese physician Terl used the method to treat 173 patients with rheumatism. In 1935, American Baker published a monograph on bee sting therapy. In 1936, China launched large-scale bee sting therapy research. In 1996, Traditional Chinese Medicine bee therapy expert You Dayun published "A Case of Paralysis Caused by Stroke Cured by Bee Sting."

[0004] Historically, bee sting therapy has been a key indication for the treatment of rheumatic and rheumatoid arthritis, and its use continues to this day. Despite this, the therapy has yet to gain widespread acceptance in modern medicine, primarily due to a lack of understanding of its active ingredients and mechanisms of action. Essentially, bee venom is a toxic mixture used by bees to protect themselves from attack. It contains numerous components with diverse physiological and toxicological effects. For example, melittin, which accounts for 50% of the venom's dry weight, is highly hemolytic, earning it the name "hemolytic peptide." Direct administration of these components to the human body, while producing therapeutic effects, inevitably results in tissue and body damage.

[0005] To further develop the long-standing tradition of bee sting therapy and enable it to serve human health through modern medical methods, we need to delve into the following questions: 1) How can we distinguish the toxic and active components in bee venom? 2) How can we identify the active components that are beneficial for treating diseases? 3) How can we elucidate the therapeutic effects and mechanisms of the active components? Although this research approach appears straightforward and clear, given the complex structures and diverse activity profiles of biomacromolecules, there are significant uncertainties regarding whether the toxic and active components in bee venom can be effectively separated, whether the active components can be retained while removing the toxic components, and whether the goal of reducing toxicity and increasing efficacy can ultimately be achieved. This research is fraught with challenges. Summary of the Invention

[0006] Bee venom is a complex toxic mixture that contains not only dozens of protease components such as phospholipase A2 and hyaluronidase, but also melittin, apamin, and mast cell degranulating peptide (Mast Cell Degranulating-peptide, Gene symbol: Mcdp; Gene ID: 406134; Protein sequence: IK C NCKRHVIKPHI C Bee venom contains more than ten oligopeptide components, including bee venom (RKICGKN), as well as a large number of non-protein components such as histamine and acids. Oligopeptide components account for 70-80% of the dry weight of bee venom, including bee venom peptide, bee venom peptide, mast cell degranulation peptide, cardiac peptide, calming peptide, tetrapin peptide (topeptide), and andurapine (andrapine). Many of these components have anti-inflammatory effects, for example: 1) Melittin (melittin hemolytic peptide), which accounts for 40-60% of the dry weight of bee venom, can promote the secretion of adrenocortical hormones by the anterior pituitary gland, leading to enhanced adrenal cortical function and exerting anti-inflammatory effects. Its anti-inflammatory activity is 100 times that of hydrocortisone, and it can inhibit the growth and reproduction of more than 20 Gram-negative and Gram-positive bacteria. It can also kill penicillin-resistant Staphylococcus aureus (a bee venom peptide variant and its application, CN201910854956; a new bee venom peptide and its application, CN202011100249). 2) Anduin, which can exert anti-inflammatory and analgesic effects by inhibiting brain prostaglandin synthase (cyclooxygenase), has an inhibitory effect 70 times that of the anti-rheumatic drug indomethacin. 3) Mast cell degranulation peptide, which accounts for 1-2.5% of the dry weight of bee venom. In the study of bee venom components, it was found that many oligopeptides exhibit diverse effects. Taking mast cell degranulation peptide as an example, on the one hand, it can promote mast cell degranulation, release histamine and 5-hydroxytryptamine, and stimulate inflammatory responses; on the other hand, studies have also found that it also has strong anti-inflammatory activity, although the mechanism of its anti-inflammatory effect is still unclear. In addition, subcutaneous injection of mast cell degranulation peptide can also increase local vascular permeability and exert a blood pressure lowering effect.

[0007] From a druggability perspective, this study focused on the druggability of oligopeptide components and identified components with anti-inflammatory properties. The results are as follows: 1) Purification and activity studies of oligopeptide components from bee venom: Using dextran gel separation technology, oligopeptide components from bee venom were first purified. Using cytotoxicity assays and a rat rheumatoid arthritis model, the biological activities of "whole bee venom" and purified oligopeptide components were compared and analyzed. The oligopeptide components retained the anti-inflammatory activity of "whole bee venom," indicating that bee venom oligopeptides are the primary active substances in bee sting therapy for rheumatic and rheumatoid arthritis. 2) Purification and preparation of oligopeptide components from bee venom: Using liquid chromatography, three major oligopeptide components from bee venom were purified to obtain melittin, apamin, and mast cell degranulation peptide. 3) Cellular pharmacology studies of the three oligopeptides: Purified apamin, mast cell degranulation peptide, and melittin were performed on cultured inflammatory fibroblast-like synoviocytes (CIA-FLS). We selected IL-1B, IL-6, and iNOS, which are highly expressed in rheumatoid joints, as cellular inflammatory markers. We analyzed the effects of apamin, mast cell degranulation peptide, and melittin on gene expression. We found that only mast cell degranulation peptide exhibited significant anti-inflammatory activity, while apamin and melittin exerted a moderate pro-inflammatory effect. 4) Animal Pharmacology of the Three Melittin Oligopeptides: Utilizing the previously established rat arthritis model, we further investigated the pharmacological effects of the three melittin oligopeptides in whole animals. Consistent with the cellular findings, mast cell degranulation peptide also exhibited strong anti-inflammatory activity in whole animals, while melittin exhibited a strong pro-inflammatory effect, with statistically significant differences in these effects. 5) Furthermore, we investigated the therapeutic effects of melittin oligopeptides on arthritis using a p-xylene-induced mouse ear swelling model and a type II collagen-induced rat arthritis model, finding that mast cell degranulation peptide exhibited a clear therapeutic effect.

[0008] Based on this work, we present the present application, which is specifically directed to the subject matter defined in the following sequentially numbered paragraphs.

[0009] 1. Use of mast cell degranulation peptide and its functional analog polypeptides, or their derivatives in the preparation of agents with anti-inflammatory effects.

[0010] 2. According to paragraph 1, it is characterized in that the functional analog polypeptide of the mast cell degranulation peptide comprises a fragment having the amino acid sequence shown in SEQ ID NO.01 (SEQ ID NO.01: IK C NCKRHVIKPHI C RKICGKN); preferably, the functional analog polypeptide of the mast cell degranulation peptide comprises a fragment having the amino acid sequence shown in SEQ ID NO.02 (SEQ ID NO.02:C NCKRHVIKPHI C RKIC).

[0011] 3. According to paragraph 1, it is characterized in that the polypeptide derivative is selected from glycopeptides, lipopeptides, deuterated peptides, protein fusion peptides, non-natural amino acid substituted polypeptides, and pharmaceutically acceptable polypeptide salts.

[0012] 4. According to paragraph 1, it is characterized in that the anti-inflammatory effect includes anti-rheumatic and rheumatoid arthritis effects.

[0013] 5. According to paragraph 1, it is characterized in that the dosage form of the agent is selected from injection, cream, ointment, patch, spray, hydrogel, and microneedle.

[0014] 6. According to paragraph 1, it is characterized in that the agent is used in combination with other anti-rheumatic and rheumatoid arthritis drugs.

[0015] 7. According to paragraph 1, it is characterized in that the polypeptide or its derivative is used in the preparation of a drug for preventing and / or treating rheumatic arthritis or rheumatoid arthritis.

[0016] In one aspect, the present invention provides the use of mast cell degranulation peptides and their functional analogs, or derivatives thereof, in the preparation of agents having anti-inflammatory effects. Preferably, the anti-inflammatory effects include anti-rheumatic and rheumatoid arthritis effects.

[0017] On the other hand, the "functional analog polypeptide of mast cell degranulation peptide" described in the present invention is a polypeptide variant formed by substituting, deleting, or adding one or more amino acids to the amino acid sequence of mast cell degranulation peptide, resulting in the same or similar biological activity as mast cell degranulation peptide. According to the Chinese invention patent "A Novel Melittin Variant and Its Application (201910854956.0)", a polypeptide variant formed by substituting, deleting, or adding one or more amino acids to the amino acid sequence of a functional polypeptide has the same function as the functional polypeptide. According to the Chinese invention patent "A Method for Preparing and Anti-tumor Application of a Conformation-Locked Melittin Anoplin Derivative (202010035278.8)", the conformation of the polypeptide is locked by forming a hydrocarbon scaffold on the side chain of the Anoplin peptide segment. This conformational locking has the same biological activity as the original polypeptide. According to the Chinese invention patent "A Mutated Melittin MEL-pep and Its Application (201711354495.8)", a melittin variant with the same activity was obtained by mutating the sequence of the melittin peptide.

[0018] On the other hand, the derivatives of the polypeptide described in the present invention include various modified forms of mast cell degranulation peptide or its functional analog polypeptide, such as: polypeptide linked to fatty acid, polypeptide linked to glycosyl, polypeptide chain covalently linked to adjuvants such as bovine serum albumin, human serum albumin, or polyethylene glycol, acylation of fluorinated amino acids, replacement of L-amino acids with D-type amino acids, N-terminal acetylated polypeptide chains, C-terminal amidated polypeptide chains, N-terminal acetylated and / or C-terminal amidated polypeptide chains, and pharmaceutically acceptable salts of polypeptides. By different modifications to the polypeptide chain, the purposes of increasing polypeptide activity, reducing toxicity, and simplifying the preparation process are achieved. According to the content of the Chinese invention patent "A kind of bee venom glycopeptide and its preparation method and use-202211213491.9", the patent obtains a type of bee venom glycopeptide by glycosylation modification of bee venom peptide, which has the activity of bee venom peptide and lower hemolysis. According to the Chinese invention patent "A lipopeptide based on melittin, its preparation method, and application - 202111061557," the invention couples a C10-18 fatty acid to the N-terminus of melittin to produce a melittin lipopeptide with improved antibacterial activity and lower hemolytic activity compared to melittin. According to the Chinese invention patent "Fusion protein of urokinase-type plasminogen activator α chain and melittin and its preparation - 200710055840.8," the invention provides a melittin fusion protein conjugated to the urokinase-type plasminogen activator α chain.

[0019] On the other hand, the dosage form of the agent provided by the present invention is selected from injections, creams, ointments, patches, sprays, hydrogels, and microneedles. According to the content of the Chinese invention patent "A bee venom peptide hydrogel and its preparation method and application-202310609572.9", the invention uses hyaluronic acid and chitosan as sustained-release carriers to develop a bee venom peptide hydrogel. According to the content of the Chinese invention patent "A soluble bee venom microneedle patch and its preparation method and application-202310248633.3", the invention discloses a soluble bee venom microneedle patch and its preparation method and application. According to the content of the Chinese invention patent "A microneedle transdermal drug delivery patch for treating rheumatoid arthritis and its preparation method-CN201910029211", the invention discloses a microneedle transdermal drug delivery patch containing bee venom for treating rheumatoid arthritis.

[0020] In another aspect, the present invention discloses a combination of an agent containing the polypeptide or derivative provided herein with other drugs having anti-rheumatic or rheumatoid arthritis effects. According to Chinese invention patent No. 202010131067.4; 111298102B, a combined antibacterial method using antimicrobial peptides and fullerenes is disclosed. This method can increase cell membrane sensitivity to antimicrobial peptides, thereby lowering the critical concentration at which antimicrobial peptides take effect and improving their effectiveness at low concentrations.

[0021] On the other hand, the present invention provides the use of the polypeptide or polypeptide derivative in the preparation of a drug for preventing and / or treating rheumatic arthritis or rheumatoid arthritis. The drug for preventing and / or treating rheumatic arthritis or rheumatoid arthritis contains an effective dose of the polypeptide or its derivative, or a pharmaceutically acceptable carrier / excipient when necessary. The pharmaceutically acceptable carrier refers to a carrier that does not interfere with the biological activity of the active ingredient, including those conventionally used in the pharmaceutical field. The pharmaceutically acceptable carrier of the present application can be solid or liquid, including pharmaceutically acceptable excipients, buffers, emulsifiers, stabilizers, preservatives, diluents, encapsulants, fillers, etc. For example, pharmaceutically acceptable buffers further include phosphates, acetates, citrates, borates, and carbonates, etc.

[0022] Throughout the description and claims, the words “comprises,” “comprising,” “containing,” and “selected from” mean “including but not limited to,” and are not intended to exclude other parts, additives, components, or steps.

[0023] Beneficial effects of the present invention

[0024] Bee venom is a complex toxic mixture, in which a large number of components have been found to have anti-inflammatory effects. But specifically in the treatment of inflammatory diseases, which component is the main active substance? There has been no clear answer. Based on a large amount of research work, this application conducted a comparative study on three main bee venom peptide substances and found that compared with bee venom peptide and bee venom peptide, mast cell degranulation peptide has outstanding anti-inflammatory effects and therapeutic effects on animal arthritis models, indicating that it is the main anti-inflammatory component of bee venom and can be used to prepare drugs for the treatment of rheumatic and rheumatoid arthritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 , the effect diagram of the separation of bee venom components by dextran gel molecular sieve.

[0026] Figure 2 , cytotoxicity test of bee venom and bee venom oligopeptides.

[0027] Figure 3, Photos of rat paws on day 15 after administration. A, saline sham treatment group; B, low-dose cervus and melon polypeptide group; C, bee venom group.

[0028] Figure 4 , The measurement results of the ankle circumference of rats. The data of the experimental group were statistically analyzed with the data of the control group as the reference. The data with significant differences are marked as: **, P < 0.01; ***, P < 0.001.

[0029] Figure 5 The results of the rat palm thickness measurements were statistically analyzed with the control group data as a reference. The data with significant differences are marked as: **, P < 0.01; ***, P < 0.001.

[0030] Figure 6 , HPLC analysis spectrum of bee venom oligopeptide.

[0031] Figure 7 , preparation spectrum of bee venom oligopeptide.

[0032] Figure 8 , mass spectrometry analysis spectrum of bee venom oligopeptide.

[0033] Figure 9 , a preliminary experiment on cell administration of bee venom oligopeptide components. The data of the experimental group were statistically analyzed with the data of the control group as a reference. Data with significant differences are marked as: **, P<0.01; ***, P<0.001.

[0034] Figure 10 The data of the experimental group were statistically analyzed with the control group as the reference. The data with significant differences are marked as: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

[0035] Figure 11 The dose-effect relationship of mast cell degranulation peptide administration was analyzed statistically with the control group data as the reference. The data with significant differences are marked as: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

[0036] Figure 12 The data of the experimental group were statistically analyzed with the control group data as reference. The data with significant differences are marked as: *, P < 0.05; **, P < 0.01.

[0037] Figure 13The data of the experimental group were statistically analyzed with the control group as the reference. The data with significant differences are marked as: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

[0038] Figure 14 The measurement results of rat palm thickness were compared. The experimental group data were statistically analyzed with the control group data as reference. The data with significant differences are marked with *, P < 0.05.

[0039] Figure 15 , The measurement results of the ankle circumference of rats. The data of the experimental group were statistically analyzed with the data of the control group as the reference. The data with significant differences are marked with: *, P < 0.05. DETAILED DESCRIPTION

[0040] The present invention will be further described below in conjunction with specific examples and accompanying drawings. It should be understood that these examples are only used to illustrate the present invention and cannot be used to limit the scope of the present invention. Without departing from the technical solutions and principles proposed in the present invention, any changes, modifications, substitutions, combinations, and simplifications made to the present invention should be equivalent replacement methods and are included in the scope of protection of the present invention. The experimental methods for which specific conditions are not specified in the following examples are generally carried out under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer.

[0041] Example 1: Purification and activity study of bee venom oligopeptide components

[0042] Bee venom is a complex toxic mixture containing dozens of proteases (phospholipase A2, hyaluronidase, etc.), more than ten oligopeptides (melittin, apamin, mast cell degranulation peptide, cardiac peptide, calming peptide, toltipine, and endurin), and a large number of non-protein components (histamine, acids, etc.), many of which have anti-inflammatory activity. To identify oligopeptides present in bee venom that have therapeutic effects on rheumatic and rheumatoid arthritis, we conducted this study.

[0043] 1. Isolation and purification of bee venom oligopeptides

[0044] Weigh a certain amount of commercial bee venom powder and place it in a centrifuge tube. Add a certain amount of purified water to the container and shake it to dissolve. Filter through a 0.22-micron filter to remove undissolved impurities to obtain a bee venom solution.

[0045] While gently shaking, add an equal volume of anhydrous ethanol dropwise to the bee venom solution. Observe the appearance of a white, flocculent precipitate. Once no new precipitate forms, transfer the centrifuge tube to a centrifuge. Centrifuge at 2000 rpm for 5 minutes at 4°C to concentrate the precipitate at the bottom of the tube. Carefully remove the liquid from the tube and dry the precipitate using a nitrogen blower. This will yield the preliminarily purified bee venom, which will be stored in a -30°C refrigerator until ready for use.

[0046] Prepare a dextran gel molecular sieve separation column and connect it to the AKTA protein analysis system. Using this device, the preliminarily purified bee venom sample was subjected to molecular sieve gel separation and the oligopeptide components were collected. The separation spectrum is shown in Figure 1 The oligopeptide components were freeze-dried to obtain bee venom oligopeptide dry powder, which was stored in a -30°C refrigerator for later use.

[0047] 2. Cytotoxicity Study

[0048] During the separation and purification of biological samples, there is a possibility that components may be lost or peptides may be denatured and inactivated due to the process. To eliminate this possibility, we used a cytotoxicity test to compare bee venom before purification (control group, complete bee venom) and bee venom oligopeptides after purification (purified group).

[0049] HEK 293T cells were cultured to the logarithmic growth phase. Cells in the logarithmic growth phase were trypsinized to prepare a single cell suspension. Cell count was performed and the cell density was adjusted to 5 × 10 4 / mL. Add 200 μL of the cell suspension to each well of a sterile 96-well plate. Place the plate in a 37°C, 5% CO2 incubator and incubate for 12 hours.

[0050] Prepare cell culture medium containing pre-purified bee venom and purified bee venom oligopeptide samples at concentrations of 0, 5, 10, 20, 50, and 100 μg / mL. Replace the culture medium with these bee venom and bee venom oligopeptide-containing solutions and continue culturing for 24 hours. Perform three replicates for each sample.

[0051] MTT assay was performed using a microplate reader at OD 490 The absorbance value of each culture well was detected at 37°C. Based on three repeated experiments, the cell inhibition rate and IC 50 Value, the result is Figure 2 shown.

[0052] Statistical analysis results showed excellent consistency between the two sets of data, indicating that the purification process did not significantly affect the biological activity of bee venom oligopeptides. The obtained bee venom oligopeptide samples retained the active ingredients of "complete bee venom" and could be used for subsequent research.

[0053] 3. Animal Pharmacology Studies on Bee Venom

[0054] The adjuvant-induced rat paw arthritis model, which mimics the symptoms of human rheumatoid arthritis to a certain extent, has been widely used as a testing platform for anti-inflammatory drugs. Using this rat rheumatoid arthritis model, we investigated the therapeutic effects of pre-purified bee venom samples on the model animals. The experimental control was a deer-melon polypeptide injection (National Medicine Standard No. H23020001, Harbin Yuheng Pharmaceutical Co., Ltd.). Deer-melon polypeptide is a sterile aqueous solution extracted from the bones of sika deer (a species of Cervidae) and melon seeds. Indications include rheumatic and rheumatoid arthritis, ankylosing spondylitis, various types of fractures, wound repair, and low back and leg pain.

[0055] Experimental animals: 40 male SD rats (SPF grade) were obtained. After one week of adaptive feeding, 6 rats were randomly selected as blank control group rats, and the rest were experimental group rats.

[0056] Rat Paw Arthritis Modeling: After adaptive feeding, the experimental group rats were treated for modeling. The rat toes and foot skin were disinfected with 75% alcohol. Then, 0.1 ml of Freund's complete adjuvant was injected intradermally into the right hind paw. On days 1, 3, 5, and 7 after Freund's complete adjuvant injection, the rats were weighed, ankle circumference and paw thickness were measured using a paw-measuring device, and movement disorders and paw licking behaviors were observed and photographed. Based on these indicators, the modeling results were evaluated, and 24 rats with successful modeling and similar weight and average paw circumference were selected for subsequent studies.

[0057] Treatment of arthritis in rats: 24 rats with successful modeling were randomly divided into 4 groups, with 6 rats in each group. The groups were treated with saline sham treatment, bee venom treatment, high-dose cervus melon polypeptide, and low-dose cervus melon polypeptide respectively.

[0058] Preparation of Bee Venom Injection: Pre-collect bee venom samples stored at -30°C. Half an hour before the experiment, weigh a predetermined amount of dry bee venom powder and add a predetermined amount of normal saline. Vortex the solution to dissolve the bee venom sample, creating a 2.5 mg / mL bee venom solution. Filter the solution through a 0.45 μm filter.

[0059] Deer and melon polypeptide injection: The polypeptide concentration of deer and melon polypeptide injection is 2mg / mL.

[0060] Arthritis rats were treated with the following medications: the bee venom group received a bee venom solution at a dose of 1.25 mg / kg body weight / day; the high-dose cervus and cucurbit peptide group received a 4 mg / kg body weight / day cervus and cucurbit peptide injection; and the low-dose cervus and cucurbit peptide group received a 2.5 mg / kg body weight / day cervus and cucurbit peptide injection. A saline sham group received the same volume of saline as the bee venom group; a blank control group received no modeling or medication. The above dosages were administered subcutaneously once daily for 15 consecutive days. The volume of fluid administered was 150-250 μL / day, depending on the animal's weight.

[0061] Assessment of Arthritis Progression: Rats were weighed daily, and the progression of paw inflammation, behavioral status, and dietary habits were observed. On days 1, 3, 7, 11, and 15 after the first dose, ankle circumference and paw thickness were measured using a toe gauge and recorded. Movement disorders and paw licking were observed and photographed. Monitoring of rat weight revealed no significant changes in the bee venom and deer melon polypeptide groups compared to the control group, indicating that bee venom administration had no effect on animal growth.

[0062] Figure 3 This is a photograph of rat paws taken 15 days after drug administration. It can be seen that the animals in the saline sham-treated group had significant swelling. In contrast, the low-dose cervus melon polypeptide group showed some relief of paw swelling, and the bee venom group showed significant relief. In the bee venom group, some of the treated areas showed ulceration, possibly due to the toxic effects of the bee venom.

[0063] The experiment was repeated three times independently, and the measured data of the ankle circumference and palm thickness of the rats were statistically analyzed and plotted ( Figure 4 、 Figure 5 The results showed that on the 7th day of modeling, the ankle circumference and palm thickness of the rats in each experimental group showed a significant increase compared with the untreated blank control group, with significant differences, indicating that the model was successful. On the 15th day after administration, the ankle circumference and palm thickness of the animals in the low-dose and high-dose cervical melon polypeptide groups were significantly decreased compared with pre-treatment, and were significantly lower than those in the saline sham treatment group, with significant differences, indicating that the positive drug has a therapeutic effect.

[0064] On day 15 after administration, the bee venom group showed a statistically significant decrease in ankle circumference and palmar thickness compared to the blank control group. These results provide preliminary evidence of the therapeutic effect of bee venom in a rat model of rheumatoid arthritis. On day 3 after administration, both ankle circumference and palmar thickness showed a transient increase, likely due to the inflammatory components of bee venom.

[0065] Example 2: Purification and preparation of bee venom oligopeptide components

[0066] Using liquid chromatography, this example establishes experimental conditions for isolating the three major oligopeptide components of bee venom (melittin, apamin, and mast cell degranulation peptide). Using these conditions, a sufficient amount of melittin, apamin, and mast cell degranulation peptide was obtained, sufficient for subsequent cell-based experiments and animal model studies.

[0067] Sample preparation: Weigh 10.0 mg of purified bee venom oligopeptide powder, add 200 μL of water to dissolve it, and filter it through a 0.22 μm filter membrane to obtain a bee venom oligopeptide solution.

[0068] HPLC analysis of bee venom oligopeptides: The mobile phase consisted of 0.1% trifluoroacetic acid (0.1% TFA) in water (A) and acetonitrile (B). The detection wavelength was 280 nm, the injection volume was 20 μL, the column temperature was 20°C, and the flow rate was 1 ml / min. The gradient elution conditions were: 0-15 min, 80%-70% A; 15-30 min, 70%-60% A; 30-45 min, 60%-50% A; 45-60 min, 50% A.

[0069] Elution spectrum Figure 6 In the separation spectrum of bee venom oligopeptides, three main peaks and several small peaks appeared, indicating that this condition can achieve the separation of the three main components of bee venom oligopeptides.

[0070] HPLC preparation of bee venom oligopeptides: A semi-preparative HPLC column was used to separate and prepare the components of bee venom oligopeptides.

[0071] The mobile phase consisted of 0.1% trifluoroacetic acid (0.1% TFA) in water (A) and acetonitrile (B). The detection wavelength was 280 nm, the injection volume was 20 μL, the column temperature was 20°C, and the flow rate was 1 ml / min. The elution program was a gradient elution: 0-15 min, 80%-70% A; 15-30 min, 70%-60% A; 30-45 min, 60%-50% A; 45-60 min, 50% A.

[0072] Elution spectrum Figure 7 As shown. During the separation process, the three main peaks were collected separately in centrifuge tubes, and the oligopeptides outside the three main peaks were collected together as "other oligopeptide components" for subsequent research. The collected components were freeze-dried and the freeze-dried powder was stored at -30°C.

[0073] Mass spectrometry identification of bee venom oligopeptide components: The separated components 1, 2, and 3 were identified by mass spectrometry and were determined to be apamin, mast cell degranulation peptide (MCD-peptide), and melittin, respectively. Figure 8 A representative spectrum for mass spectrometry identification.

[0074] Example 3: Cellular Pharmacology Study of Three Bee Venom Oligopeptides

[0075] Using in vitro cultured cells, this example conducted cellular pharmacology studies on the obtained melittin, mast cell degranulation peptide, and melittin. The experimental protocol and conditions are as follows:

[0076] Cell Model: CIA-FLS cells are inflammatory fibroblast-like synoviocytes isolated from adjuvant-induced arthritis sites in rats. They are cultured in 90% RPMI-1640 + 10% FBS (containing glutamine) at 37°C and 5% CO2. Under normal culture conditions, these cells express inflammatory cell markers.

[0077] Cell administration: CIA-FLS cells were cultured to the logarithmic growth phase and digested with trypsin to prepare a cell concentration of 2×10 5 CIA-FLS cells were seeded into 6-well cell culture plates at a volume of 2 mL per well. After culturing for 24 hours, the cell culture medium was replaced with medium containing a specific drug concentration for drug treatment. Three replicate wells were used for each drug treatment.

[0078] Gene Expression Study: Three hours after treatment, cells were harvested. Total RNA was extracted and reverse transcribed to obtain cDNA according to standard protocols. Real-time quantitative PCR was performed using cDNA as a template to analyze changes in the expression levels of three cytokines: IL-1B, IL-6, and iNOS. The GADPH gene was used as an internal control. IL-1B, IL-6, and iNOS are highly expressed in rheumatoid joints and were selected as inflammatory markers in this study.

[0079] The amplification primers for IL-1B, IL-6, and iNOS genes are:

[0080] GAPDH-FP (SEQ ID NO. 11), 5'-GGCACAGTCAAGGCTGAGAATG;

[0081] GADPH-RP (SEQ ID NO. 12), 5'-ATGGTGGTGAAAGACGCCAGTA;

[0082] rIL-1B-FP (SEQ ID NO. 13), 5'-CTTGTTTCATTCTGAGCCTCCTC;

[0083] rIL-1B-RP (SEQ ID NO. 14), 5'-ATATGTCGGGCTGGTTCCAC;

[0084] rIL-6-FP (SEQ ID NO. 15), 5'-ATTGTATGAACAGCGATGATGC;

[0085] rIL-6-RP (SEQ ID NO. 16), 5'-AGAAACGGAACTCCAGAAGACC;

[0086] riNOS-FP (SEQ ID NO. 17), 5'-GGATATCTTCGGTGCGGTCTT;

[0087] riNOS-RP (SEQ ID NO. 18), 5'-CTGTAACTCTTCTGGGTGTCAGA.

[0088] Real-time fluorescence quantitative PCR amplification: pre-denaturation at 95°C for 3 minutes, followed by 44 cycles of amplification, the cycle conditions are 95°C, 15s→54-60°C, 30s→72°C, 30s. △△ The Ct method was used to calculate the relative expression of the target gene relative to the internal reference gene.

[0089] Preliminary cellular pharmacology experiment of bee venom oligopeptide components: According to the above research plan, we conducted a preliminary analysis of the pharmacological effects of bee venom peptides, and the control drugs were deer melon polypeptide and hyaluronic acid. The dosing regimen and dosing concentration were: CTRL (blank control), no drug intervention; BVP (bee venom oligopeptide component group, bee venom oligopeptide mixture prepared by HPLC purification), drug treatment concentration was 2ug / mL; CCP L (deer melon polypeptide low-dose group), drug treatment concentration was 10μg / mL; CCP H (deer melon polypeptide high-dose group), drug treatment concentration was 40μg / mL; HA (hyaluronic acid group, hyaluronic acid molecular weight range 100-600kDa), drug treatment concentration was 100μg / mL. The experiment was repeated three times independently, and the experimental results were statistically analyzed and plotted, as shown below. Figure 9 shown.

[0090] Figure 9 The experimental results shown are as follows: 1) Hyaluronic acid had no effect in the cultured cell study; 2) Only in the high-dose group of deer melon polypeptide was the expression of inflammatory factors downregulated to a certain extent; 3) Under the experimental concentration conditions, the administration of bee venom oligopeptide components significantly reduced the expression levels of three cell markers, indicating that the expression of inflammatory genes was inhibited at the cellular level.

[0091] Dose-effect relationship of dexamethasone administration: Dexamethasone is a commonly used anti-inflammatory drug and was used as a positive control drug in this study. This experiment studied the anti-inflammatory activity of different concentrations of dexamethasone. The dosing regimen and concentration were as follows: CTRL (blank control group), no drug intervention; DXMS1 (low-dose dexamethasone group), drug treatment concentration was 1μg / mL; DXMS 10 (medium-dose dexamethasone group), drug treatment concentration was 10μg / mL; DXMS100 (high-dose dexamethasone group), drug treatment concentration was 100μg / mL. The experiment was repeated three times independently, and the experimental results were statistically analyzed and plotted, as shown below. Figure 10 shown.

[0092] Figure 10 The results showed that dexamethasone at two concentrations of 1.0μg / ml and 10.0μg / ml caused a decrease in the three cell markers, demonstrating good anti-inflammatory activity.

[0093] Dose-effect relationship of mast cell degranulation peptide administration: This experiment studied the anti-inflammatory activity of mast cell degranulation peptide at different concentrations. The dosing regimen and dosing concentration were as follows: CTRL (blank control group), no drug intervention; MP-0.5 (mast cell degranulation peptide 0.5 microgram group), drug treatment concentration was 0.5 μg / mL; MP-1 (mast cell degranulation peptide 1.0 microgram group), drug treatment concentration was 1.0 μg / mL; MP-1.5 (mast cell degranulation peptide 1.5 microgram group), drug treatment concentration was 1.5 μg / mL; MP-2 (mast cell degranulation peptide 2.0 microgram group), drug treatment concentration was 2.0 μg / mL; MP-2.5 (mast cell degranulation peptide 2.5 microgram group), drug treatment concentration was 2.5 μg / mL; CCP (deer melon polypeptide group), drug treatment concentration was 40 μg / mL; DXMS (dexamethasone group), drug treatment concentration was 10 μg / mL. The experiment was repeated three times independently, and the experimental results were statistically analyzed and plotted, as shown in the following figure. Figure 11 shown.

[0094] Figure 11 The results showed that the administration of mast cell degranulation peptide could significantly inhibit the expression of inflammatory cytokines, indicating that it played an anti-inflammatory role at the culture level.

[0095] Dose-effect relationship of apamin: This study investigated the anti-inflammatory activity of apamin at different concentrations. The dosing schedule and concentrations were as follows: CTRL (blank control group), no drug intervention; AP-0.5 (0.5 μg apamin group), treatment with a drug concentration of 0.5 μg / mL; AP-1 (1.0 μg apamin group), treatment with a drug concentration of 1.0 μg / mL; AP-1.5 (1.5 μg apamin group), treatment with a drug concentration of 1.5 μg / mL; AP-2 (2.0 μg apamin group), treatment with a drug concentration of 2.0 μg / mL; AP-2.5 (2.5 μg apamin group), treatment with a drug concentration of 2.5 μg / mL; CCP (deer melon polypeptide), treatment with a drug concentration of 40 μg / mL; and DXMS (dexamethasone), treatment with a drug concentration of 10 μg / mL. The experiment was repeated three times independently, and the results were statistically analyzed and plotted. Figure 12 shown.

[0096] Figure 12 Results showed that when apamin was administered at a concentration of 2.0 μg / mL or above, the expression of inflammatory cytokines in the iNOS and iL-6 groups increased significantly, indicating that high-concentration administration has a pro-inflammatory effect. The expression level of the iL-1B cytokine remained stable at different administration concentrations.

[0097] Dose-effect relationship of bee venom peptide administration: This experiment studied the anti-inflammatory activity of bee venom peptide at different concentrations. The dosing regimen and concentration were: CTRL (blank control group), no drug intervention; ML-0.5 (0.5 microgram bee venom peptide group), drug treatment concentration was 0.5 μg / mL; ML-1 (1.0 microgram bee venom peptide group), drug treatment concentration was 1.0 μg / mL; ML-1.5 (1.5 microgram bee venom peptide group), drug treatment concentration was 1.5 μg / mL; ML-2 (2.0 microgram bee venom peptide group), drug treatment concentration was 2.0 μg / mL; ML-2.5 (2.5 microgram bee venom peptide group), drug treatment concentration was 2.5 μg / mL; CCP (deer melon polypeptide), drug treatment concentration was 40 μg / mL; DXMS (dexamethasone), drug treatment concentration was 10 μg / mL. The experiment was repeated three times independently, and the experimental results were statistically analyzed and plotted, as shown below. Figure 13 shown.

[0098] Figure 13 The results showed that when melittin was administered at a low concentration of 0.5 μg / mL, it significantly promoted the expression of three cytokines; further increasing the dosage to 2.5 μg / mL further enhanced the pro-inflammatory effect. This indicates that at the cellular level, melittin is the pro-inflammatory component of bee venom oligopeptides.

[0099] Using cultured cell experiments, this example investigated the anti-inflammatory effects of three major bee venom oligopeptides at the molecular level. Among them, only mast cell degranulation peptide exhibited significant anti-inflammatory activity, while apamin and melittin exhibited certain pro-inflammatory effects.

[0100] Example 4: Study on the anti-inflammatory effects of three bee venom oligopeptides

[0101] In this example, we used the rat arthritis model established in Example 1 to further study the pharmacological effects of three bee venom oligopeptides at the whole animal level.

[0102] The research plan and experimental process are described in detail in Example 1.

[0103] Thirty rats with successfully established arthritis models were obtained according to the method described in Example 1 and randomly divided into five groups of six rats each. The groups were treated with mast cell degranulation peptide, apamin, melittin, a trace component (a mixture of oligopeptide components of the melittin component other than mast cell degranulation peptide, apamin, and melittin), and a saline sham treatment group.

[0104] The dosing regimen and dosage for the model rats were as follows: MDP (1.25 mg / kg body weight); Apamin (1.25 mg / kg body weight); Melittin (1.25 mg / kg body weight); and the trace component group (1.25 mg / kg body weight). A sham saline treatment group received the same volume of saline as the MDP group. The volume of fluid administered by injection was 150-250 μL / day, depending on the animal's body weight.

[0105] The drug preparation, administration, and validation evaluation were performed according to the method described in Example 1. The experiment was repeated three times independently, and the experimental results were statistically analyzed and plotted ( Figure 14 、 Figure 15 ).

[0106] The experimental results showed that after 15 days of administration, compared with the saline sham treatment group, mast cell degranulation peptide showed stronger anti-inflammatory activity, while melittin showed stronger pro-inflammatory effects. These differences were statistically significant (*, P < 0.05). Melittin had no significant anti-inflammatory effect. Comparison of studies in animals and cultured cells revealed good consistency in the anti-inflammatory effects of the three peptides.

[0107] Example 5 Effects of three bee venom oligopeptides on xylene-induced ear swelling in mice

[0108] In this example, we used the p-xylene-induced mouse ear swelling model to study the therapeutic effects of three bee venom oligopeptides.

[0109] Sixty KM mice (half male and half female) were obtained and randomly divided into five groups of 12 mice each. The groups were treated with mast cell degranulation peptide, apamin, melittin, a trace component (a mixture of oligopeptides from the melittin component excluding mast cell degranulation peptide, apamin, and melittin), and a saline sham treatment group.

[0110] The model mice received the following dosage and administration schedule: the MCP-treated group received 0.38 mg / kg body weight / day; the Apamin-treated group received 0.38 mg / kg body weight / day; the Melittin-treated group received 0.38 mg / kg body weight / day; the trace component-treated group received 0.38 mg / kg body weight / day; and the saline sham-treated group received the same volume of saline as the MCP-treated group. The drug was administered by bilateral injection into the base of the left and right ears once daily for 7 consecutive days.

[0111] One hour after the last dose, 0.04 mL of xylene was applied to both sides of the right ear of mice in each treatment group to induce a model of ear swelling. Thirty minutes after the xylene application, the animals were sacrificed. The left and right ear pieces were punched out at the corresponding locations and accurately weighed. The swelling rate of the right ear piece was calculated using the left ear piece as a control. This was calculated using the following formula: Swelling rate = ((right ear weight - left ear weight) / left ear weight) × 100%.

[0112] The test data were converted into swelling rate and then statistically processed, and variance analysis was performed between groups. The results are shown in Table 1.

[0113] Table 1 Effects of three bee venom oligopeptides on xylene-induced ear swelling in mice (n=12)

[0114]

[0115] Note: Compared with the saline sham-treated group, *P < 0.05, **P < 0.01.

[0116] The experimental results showed that compared with the saline sham treatment group, the administration of mast cell degranulation peptide showed stronger anti-inflammatory activity (P < 0.01), and the administration of bee venom peptide showed stronger pro-inflammatory effect (P < 0.05), and these effects were statistically significantly different.

[0117] Example 6: Therapeutic effects of three bee venom oligopeptides on collagen-induced arthritis in rats

[0118] Rheumatoid arthritis is an autoimmune disease characterized by peripheral joint involvement. Its pathological manifestations include joint inflammation and synovial angiogenesis, which in turn affect cartilage lesions, leading to joint deformity and loss of function. Type II collagen-induced arthritis (CIA) in rats is a recognized research model. Its pathogenesis closely resembles that of human rheumatoid arthritis and has been widely used in related research. For details, please refer to the Chinese invention patents "A pharmaceutical composition for preventing and treating rheumatoid arthritis and its preparation method (CN201810571496)", "Use of a polypeptide in the preparation of a drug for treating or preventing rheumatoid arthritis (CN201310729055)", "Use of a polypeptide in the preparation of a drug for treating or preventing rheumatoid arthritis (CN201410319902)", and "Use of a traditional Chinese medicine composition containing scorpions as a drug for treating bone erosion in rheumatoid arthritis (CN201910499132)".

[0119] Using the bovine type II collagen-induced arthritis model in rats, this example further studied the therapeutic effects of three bee venom oligopeptides on model animals.

[0120] Rat Arthritis Modeling: Following the method reported in the literature, a certain amount of bovine type II collagen (2 mg / mL) was mixed with an equal volume of complete Freund's adjuvant and ground to prepare a collagen emulsion. The collagen emulsion was injected subcutaneously at a dose of 0.2 mL / rat 2 cm from the base of the rat's tail. On the 7th day after injection, a secondary immunization was performed with an equal volume of a mixed emulsion of bovine type II collagen and incomplete Freund's adjuvant injected 3 cm from the base of the rat's tail. Each animal received an injection dose of 0.1 mL of emulsion. No treatment was given to the blank control group.

[0121] The rats' arthritis index (AI) was assessed the next day after the first immunization with bovine type II collagen. The scoring criteria are as follows.

[0122] The arthritis index scoring criteria are as follows: 0 points, normal joint condition; 1 point, obvious redness and swelling of the ankle or knee joint, or close to redness and swelling, and the redness and swelling spread to the fingertips; 2 points, severe redness and swelling of the ankle or knee joint; 3 points, severe redness and swelling of the entire sole of the foot including the fingertips; 4 points, unable to bear weight.

[0123] Wistar male rats were obtained. Ten rats were taken as a blank control group and did not receive any treatment. The other rats were injected with collagen emulsion for modeling according to the above scheme. After the second immunization, 50 model rats with an arthritis index between 6 and 12 were selected and divided into 5 groups of 10 rats each according to the arthritis index, namely: a mast cell degranulation peptide administration group, a bee venom peptide administration group, a bee venom peptide administration group, a trace component administration group (a mixture of other oligopeptide components in the bee venom peptide component except mast cell degranulation peptide, bee venom peptide, and bee venom peptide), and a saline sham treatment group.

[0124] The model rats received the following dosing schedule: a blank control group received no treatment; a mast cell degranulation peptide group received a dose of 1.25 mg / kg body weight; a melittin group received a dose of 1.25 mg / kg body weight; a melittin group received a dose of 1.25 mg / kg body weight; a trace component group received a dose of 1.25 mg / kg body weight; and a saline sham group received the same volume of saline as the mast cell degranulation peptide group. Subcutaneous injections were administered at the ankle, knee, sole, and toe of the affected area at the above doses once daily for 15 consecutive days. The volume of fluid administered ranged from 250 to 450 μL / day, depending on the animal's weight.

[0125] Starting from the day of administration, rats were weighed daily as described in Example 1, and their behavioral and dietary habits were observed. On the day of administration (day 1) and on days 4, 7, 10, 13, and 16 after administration, the degree of swelling in the rats' toes was measured using a toe volume meter and recorded. The rats' arthritis index was evaluated according to the Arthritis Index Scoring Criteria.

[0126] Synovial Tissue Pathology: On day 16 after administration, after measurement of paw swelling and assessment of the arthritis index, the rats were sacrificed and the knee joints removed. The knee joint cavity was opened, and the synovial tissue was excised and fixed in 4% paraformaldehyde. Following a protocol previously reported, synovial tissue sections were prepared through fixation, dehydration, clearing, wax impregnation, embedding, sectioning, and staining. Morphological changes in the synovial tissue were analyzed under a light microscope.

[0127] Data analysis: SPSS18 statistical software was used to perform one-way analysis of variance on the obtained data, and t-test was used for comparison between groups.

[0128] The experimental results are as follows.

[0129] 1. General observations. a) Blank control group: The rats grew normally, had smooth fur, were in good spirits, and showed no abnormalities. b) Normal saline sham treatment group: After the initial immunization, the rats developed redness and swelling of the tail on the second and third days, and the symptoms gradually worsened. After the second day after the booster immunization, the rats' tails gradually became congested and ulcerated, and began to scab on the sixth day. Subsequently, the rats began to become ill, with redness and swelling of the ankles, knees, and toes, and thickening of the foot pads. The symptoms worsened until joint function and movement became impaired and crawling became difficult. During this pathological process, the rats' food intake gradually decreased and their weight decreased. c) Mast cell degranulation peptide group: After administration, the above symptoms of the rats were alleviated to varying degrees. Ten days after administration, the rats' activities basically returned to normal, their food intake increased significantly compared to the previous period, and their weight rebounded. d) Melittin group: Melittin administration did not significantly improve the symptoms of the model mice. e) In the melittin group, melittin administration significantly worsened or accelerated the pathological process in the rats, with worsening symptoms in the ankles, knees, toes, and footpads. In the later stages of administration, some rats developed deformed toes, difficulty moving and becoming unable to bear weight, dry hair, and significant weight loss. f) In the trace component administration group, administration had no significant effect on the symptoms of the rats.

[0130] 2. Assessment of rat arthritis index:

[0131] On the day of administration (day 1) and on days 4, 7, 10, 13, and 16 after administration, the arthritis index of the ankle joint, knee joint, etc. was evaluated according to the Arthritis Index Scoring Standard. The results are shown in Table 2.

[0132] Table 2. Arthritis index of rats

[0133]

[0134]

[0135] Note: The significance of the differences in the data of the drug-treated animals was calculated with the saline sham-treated group as the reference; the data with significant differences are marked as: *, P<0.05; **, P<0.01; ***, P<0.001.

[0136] Compared with the blank control group, the arthritis index in the saline sham-treated group was significantly increased, with significant differences observed at all test time points (P < 0.01), indicating successful model establishment. Compared with the saline sham-treated group, the arthritis index in the mast cell degranulation peptide-treated group was significantly reduced, indicating relief of arthritis symptoms and demonstrating the therapeutic effect of mast cell degranulation peptide on rat joints. In the melittin-treated group, the arthritis index remained elevated throughout the treatment period and, in the later stages of treatment, was significantly higher than that in the saline sham-treated group.

[0137] 3. Effect on rat foot swelling:

[0138] On the day of administration (day 1) and on days 4, 7, 10, 13, and 16 after administration, the paw volume (μl) was measured and recorded. The significance of the differences in the data of the animals in each administration group was calculated using the saline sham treatment group as a reference. The results are shown in Table 3.

[0139] Table 3. Measurement results of rat toe swelling

[0140]

[0141]

[0142] Note: The significance of the differences in the data of the drug-treated animals was calculated with the saline sham-treated group as the reference; the data with significant differences are marked as: *, P<0.05; **, P<0.01; ***, P<0.001.

[0143] Compared with the blank control group, the degree of toe swelling in the saline sham treatment group was significantly increased, and significant differences were found at all test time points (P<0.01), indicating that the model was successfully established. Compared with the saline sham treatment group, the toe swelling in the mast cell degranulation peptide administration group was significantly alleviated, and in the later period of administration, it was basically close to the measured value of the blank control group animals, reflecting the therapeutic effect of mast cell degranulation peptide on rat joints. In the bee venom peptide administration group, the toe swelling of the animals was significantly higher than that in the saline sham treatment group, showing a strong pro-inflammatory effect. The administration of bee venom peptide and trace components had little effect on the symptoms of rats. These are consistent with the previous experimental results.

[0144] 4. Effects on rat synovial tissue:

[0145] Compared with the blank control group, the synovial tissue in the saline-treated rats showed significant hyperplasia, covered with multiple layers of synovial cells, accompanied by inflammatory cell infiltration, vascular proliferation, and dilated congestion. Histopathological observations in the mast cell degranulation peptide group were similar to those in the blank control group. The synovial tissue in most animals had essentially returned to normal, while a few were still in the process of recovery, with a small amount of inflammatory cell infiltration observed. Symptoms in the melittin group were more severe than in the saline-treated group, with more pronounced synovial tissue and vascular proliferation and more severe inflammatory cell infiltration. Melittin and the trace component administration groups had no significant effect on symptoms, and the extent of tissue damage in the animals was generally consistent with that in the saline-treated group.

[0146] Conclusion: This example investigated the therapeutic effects of collagen-induced arthritis in rats. The data showed that, among the three major bee venom oligopeptides, mast cell degranulation peptide had a significant therapeutic effect on rat arthritis, improving the pathological state of the rats in multiple aspects. In contrast, administration of melittin resulted in a worsening of symptoms.

[0147] Example 7: Study on the therapeutic effects of “complete bee venom” and mast cell degranulation peptide

[0148] According to the protocol described in Example 6, a bovine type II collagen-induced arthritis model in rats was established, and the therapeutic effects of "complete bee venom" (unpurified bee venom) and mast cell degranulation peptide on the model animals were compared.

[0149] Wistar male rats were obtained. Six of these rats served as a blank control group and received no treatment. The remaining rats were injected with collagen emulsion according to the above protocol to establish the model. After the second immunization, 102 model rats with arthritis index between 6 and 12 were selected and divided into 17 groups according to the arthritis index, with 6 rats in each group, namely: mast cell degranulation peptide 0.1 mg group, mast cell degranulation peptide 0.2 mg group, mast cell degranulation peptide 0.5 mg group, mast cell degranulation peptide 0.75 mg group, mast cell degranulation peptide 1.0 mg group, mast cell degranulation peptide 1.25 mg group, mast cell degranulation peptide 2.0 mg group, mast cell degranulation peptide 4.0 mg group, complete bee venom 0.1 mg group, complete bee venom 0.2 mg group, complete bee venom 0.5 mg group, complete bee venom 0.75 mg group, complete bee venom 1.0 mg group, complete bee venom 1.25 mg group, complete bee venom 2.0 mg group, complete bee venom 4.0 mg group, and saline sham treatment group.

[0150] The dosage and administration schedule of the model rats were as follows: blank control group, no treatment; mast cell degranulation peptide 0.1 mg group, the dosage was 0.1 mg / kg body weight; mast cell degranulation peptide 0.2 mg group, the dosage was 0.2 mg / kg body weight; mast cell degranulation peptide 0.5 mg group, the dosage was 0.5 mg / kg body weight; mast cell degranulation peptide 0.75 mg group, the dosage was 0.75 mg / kg body weight; mast cell degranulation peptide 1.0 mg group, the dosage was 1.0 mg / kg body weight; mast cell degranulation peptide 1.25 mg group, the dosage was 1.25 mg / kg body weight; mast cell degranulation peptide 2.0 mg group, the dosage was 2.0 mg / kg body weight; mast cell degranulation peptide 4.0 mg group, the dosage was The 0.1 mg whole bee venom group received a dose of 0.1 mg / kg body weight; the 0.2 mg whole bee venom group received a dose of 0.2 mg / kg body weight; the 0.5 mg whole bee venom group received a dose of 0.5 mg / kg body weight; the 0.75 mg whole bee venom group received a dose of 0.75 mg / kg body weight; the 1.0 mg whole bee venom group received a dose of 1.0 mg / kg body weight; the 1.25 mg whole bee venom group received a dose of 1.25 mg / kg body weight; the 2.0 mg whole bee venom group received a dose of 2.0 mg / kg body weight; and the 4.0 mg whole bee venom group received a dose of 4.0 mg / kg body weight. The saline sham group received the same volume of saline as the mast cell degranulation peptide group. The above-mentioned doses were administered subcutaneously once a day for 15 consecutive days at the affected areas of the ankles, knees, soles, and toes. The volume of fluid administered by injection was 250-450 uL / day, depending on the animal's body weight.

[0151] The therapeutic effects of whole bee venom and mast cell degranulation peptide on model animals were evaluated according to the method described in Example 1. The experimental results are as follows.

[0152] Table 4. Arthritis index of rats

[0153]

[0154]

[0155]

[0156]

[0157] Note: The significance of the differences in the data of the drug-treated animals was calculated with the saline sham-treated group as the reference; the data with significant differences are marked as: *, P<0.05; **, P<0.01; ***, P<0.001.

[0158] Table 5. Measurement results of rat toe swelling

[0159]

[0160]

[0161]

[0162] Note: The significance of the differences in the data of the drug-treated animals was calculated with the saline sham-treated group as the reference; the data with significant differences are marked as: *, P<0.05; **, P<0.01; ***, P<0.001.

[0163] Conclusion: Using the rat arthritis model induced by bovine type II collagen, this example compared the therapeutic effects of "complete bee venom" and mast cell degranulation peptide on rat arthritis. Combined with the general observation of the animal's condition during the treatment process, the evaluation of the arthritis index (Table 4), and the measurement results of the rat toe swelling (Table 5), it was found that: 1) The rats in the blank control group grew normally and were in good mental state. Judging from the arthritis index and toe measurements, there were no arthritis-related symptoms and toe swelling. 2) The situation of the animals in the saline sham treatment group was basically consistent with the observations in Example 6. After the initial immunization, the rats developed redness and swelling of the tail. After the booster immunization, the symptoms worsened, and the tail gradually became congested and ulcerated, accompanied by swelling of the ankle joint, knee joint, and toes and thickening of the foot pad, until joint functional movement disorders occurred. 3) In the mast cell degranulation peptide treatment groups with different doses, the therapeutic effect was correlated with the dosage. In the low-dose groups (0.1 mg and 0.2 mg mast cell degranulation peptides), the animals' symptoms did not improve significantly. As the treatment dose increased, the therapeutic effect gradually became apparent, and in the high-dose group, the symptom improvement reached a plateau. 4) In the different doses of complete bee venom treatment groups, the therapeutic effect and toxicity showed a certain additive effect. In the low-dose groups (0.1 mg, 0.2 mg, and 0.5 mg complete bee venom), the therapeutic effect was not obvious; in the high-dose groups (2.0 mg and 4.0 mg complete bee venom), the toxicity was very significant. Only in the medium-dose groups (0.75 mg, 1.0 mg, and 1.25 mg complete bee venom) did a certain therapeutic effect appear in the later stages of treatment.

[0164] From the data of this example, mast cell degranulation peptide and "complete bee venom" showed different therapeutic effects on arthritis. The purified mast cell degranulation peptide was used to treat arthritis in rats, completely eliminating the toxic effects of bee venom.

Claims

1. Use of mast cell degranulation peptide and its functional analog polypeptides, or their derivatives in the preparation of agents with anti-inflammatory effects.

2. According to claim 1, it is characterized in that The functional analog polypeptide of the mast cell degranulation peptide comprises a polypeptide having SEQ ID NO. 01 (SEQ ID NO.01: IKCNCKRHVIKPHICRKICGKN); preferably, the functional analog polypeptide of the mast cell degranulation peptide comprises a fragment having the amino acid sequence shown in SEQ ID NO.02 (SEQ ID NO.02: CNCKRHVIKPHICRKIC).

3. According to claim 1, it is characterized in that The polypeptide derivative is selected from glycopeptides, lipopeptides, deuterated peptides, protein fusion peptides, non-natural amino acid substituted polypeptides, and pharmaceutically acceptable polypeptide salts.

4. According to claim 1, it is characterized in that The anti-inflammatory effect includes anti-rheumatic and rheumatoid arthritis effects.

5. According to claim 1, it is characterized in that The dosage form of the agent is selected from injection, cream, gel, ointment, patch, spray, hydrogel, and microneedle.

6. According to claim 1, it is characterized in that The agent is used in combination with other anti-rheumatic and rheumatoid arthritis drugs.

7. According to claim 1, it is characterized in that The polypeptide or its derivatives are used in the preparation of drugs for preventing and / or treating rheumatic arthritis and rheumatoid arthritis.

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