Application of indole-3-formaldehyde in preparation of preparation for preventing or treating rheumatoid arthritis

Abstract

The invention belongs to the technical field of medicines, and particularly relates to an application of indole-3-formaldehyde in prevention or treatment of rheumatoid arthritis, and the indole-3-formaldehyde has no side effects such as kidney damage, hepatotoxicity, gastrointestinal reaction and the like in treatment of inflammatory reactions of mice with collagen-induced rheumatoid arthritis. In addition, the indole-3-formaldehyde provided by the invention also has the effect of protecting the intestinal mucosal barrier. The indole-3-formaldehyde is an endogenous metabolite of a human body, and is safe to organisms; and secondly, the indole-3-formaldehyde can be combined with probiotics such as bifidobacterium, lactobacillus casei and the like and prebiotics such as fructo-oligosaccharide, galactooligosaccharide, inulin and the like for use, so that the conflict emotion of a patient to the medicine is reduced, and the indole-3-formaldehyde has a good application prospect in clinic.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to an application of indole-3-carboxaldehyde in preparing a preparation for preventing or treating rheumatoid arthritis. Background Art

[0002] Rheumatoid arthritis, also known as rheumatoid arthritis, or RA for short, is an inflammatory, chronic, systemic autoimmune disease with synovial inflammation as the main pathological manifestation. From an immunological perspective, the production of autoantibodies and the presence of effector T cells in the blood and synovial structures are significant features of rheumatoid arthritis, and rheumatoid factor and anti-citrullinated protein antibodies are the main indicators of RA. From a histological perspective, rheumatoid masses are the hallmarks of rheumatoid arthritis, accompanied by synovial tissue hyperplasia, neovascularization, and infiltration by activated heterogeneous inflammatory cells. Activated osteoclasts and chondrocytes play a direct role in the development of joint structural damage. Cytokines are also involved in the interaction between cells in the inflammatory network.

[0003] One hypothesis regarding the pathogenesis of rheumatoid arthritis is that the disease begins at mucosal sites as a result of the interaction of the mucosal immune system with an abnormal local microbiota, which then involves the synovial joints. Alterations in the composition of the intestinal microbiota in patients with preclinical and established RA suggest that intestinal dysbiosis plays a role in the development and persistence of RA.

[0004] The drugs currently used to treat rheumatoid arthritis mainly include nonsteroidal anti-inflammatory drugs, disease-modifying antirheumatic drugs, biologics, and glucocorticoids. In addition to drug therapy, stem cell transplantation, immune purification therapy, gene therapy, and surgical therapy are all used in the treatment of RA. However, the clinical phenotype of RA patients is heterogeneous, and the treatment effects are not the same. In addition, drugs have different side effects such as kidney damage, liver toxicity, and gastrointestinal reactions. In addition, RA-related complications increase the clinical demand for adverse prognostic factors and personalized treatment. Therefore, there is an urgent need to provide a new strategy for the treatment of rheumatoid arthritis. Summary of the invention

[0005] The liver and kidney toxicity and gastrointestinal reactions of the drugs for treating rheumatoid arthritis in the prior art have not been solved. The present invention provides an application of indole-3-carboxaldehyde in the preparation of a preparation for preventing or treating rheumatoid arthritis.

[0006] The technical solution adopted by the present invention is:

[0007] The invention provides an application of indole-3-carboxaldehyde in preparing a preparation for preventing or treating rheumatoid arthritis.

[0008] Preferably, the preparation uses indole-3-carboxaldehyde as an active ingredient, supplemented with probiotics, prebiotics and pharmaceutically acceptable excipients.

[0009] Preferably, the probiotics include at least one of Bifidobacterium and Lactobacillus casei.

[0010] Preferably, the prebiotics include at least one of fructooligosaccharides, galacto-oligosaccharides and inulin.

[0011] Preferably, the pharmaceutically acceptable excipient is one or more of a diluent, a disintegrant, a precipitation inhibitor, a glidant, a binder, a dispersant, a suspending agent, an isotonic agent, a thickener, an emulsifier, a preservative and a stabilizer.

[0012] Preferably, the diluent includes any one of starch, lactose, sucrose and mannitol.

[0013] Preferably, the disintegrant includes at least one of starch, microcrystalline cellulose and low-substituted hydroxypropyl cellulose.

[0014] Preferably, the precipitation inhibitor comprises any one of sodium lauryl sulfate, Tween-80, polyvinyl pyrrolidone and hydroxypropyl methylcellulose.

[0015] Preferably, the glidant includes any one of cationic polyacrylamide, polydiallyldimethylammonium chloride and cationic starch.

[0016] Preferably, the binder comprises any one of starch slurry, hydroxypropyl methylcellulose and povidone.

[0017] Preferably, the dispersant includes any one of sodium lauryl sulfate, polyvinyl pyrrolidone and sodium carboxymethyl cellulose.

[0018] Preferably, the suspending agent comprises any one of gum arabic, gum tragacanth, sodium carboxymethylcellulose and hydroxypropyl methylcellulose.

[0019] Preferably, the isotonic agent includes any one of sodium chloride, glucose and mannitol.

[0020] Preferably, the thickener includes any one of gum arabic, xanthan gum and sodium carboxymethyl cellulose.

[0021] Preferably, the emulsifier includes any one of sodium lauryl sulfate, benzalkonium chloride and sorbitan fatty acid.

[0022] Preferably, the preservative includes any one of benzoic acid, sorbic acid, methyl paraben and benzalkonium bromide.

[0023] Preferably, the stabilizer comprises any one of sodium sulfite, sodium bisulfite, tocopherol and disodium edetate.

[0024] Preferably, the active ingredient further comprises at least one of non-steroidal anti-inflammatory drugs, glucocorticoids, sodium hyaluronate, medical chitosan and growth factors.

[0025] Preferably, the non-steroidal anti-inflammatory drug comprises at least one of aspirin, ibuprofen, diclofenac and celecoxib.

[0026] Preferably, the glucocorticoid comprises at least one of prednisone and methylprednisolone.

[0027] Preferably, the content of indole-3-carboxaldehyde in the preparation is not less than 10 mg / mL.

[0028] Preferably, the acceptable dosage form of the preparation includes one of tablets, capsules, granules, injections, pills, powders, pastes and oral liquids.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The present invention provides an application of indole-3-carboxaldehyde in the preparation of a preparation for preventing or treating rheumatoid arthritis. The indole-3-carboxaldehyde is used to treat the inflammatory response of mice with collagen-induced rheumatoid arthritis, and no side effects such as kidney damage, liver toxicity and gastrointestinal reactions are found. In addition, the indole-3-carboxaldehyde provided by the present invention also has the effect of protecting the intestinal mucosal barrier. Indole-3-carboxaldehyde is an endogenous metabolite of the human body and is safe for organisms; secondly, indole-3-carboxaldehyde can be used in combination with probiotics such as Bifidobacterium and Lactobacillus casei, as well as prebiotics such as oligofructose, oligogalactose, and inulin, to reduce patients' resistance to drugs, and has good application prospects in clinical practice.

[0031] For humans, intestinal flora can further affect human physiological changes by secreting metabolites. At the same time, in the human tryptophan metabolism, the kynurenine metabolic pathway is the main metabolic pathway of the human body, accounting for about 95% of the total metabolic pathway, while the remaining part, about 5%, is metabolized through the indole pathway of the intestinal flora to produce the final specific product indole-3-formaldehyde, that is, Indole-3-aldehyde, referred to as I3A. In the human body, I3A can only be produced through the action of intestinal flora and is a unique metabolite produced by intestinal flora. Like indole, I3A can act as a metabolite and signal molecule, and can act as an agonist of aromatic hydrocarbon receptors to regulate epithelial barrier function. And I3A can regulate the development and growth of intraepithelial lymphocytes and innate lymphocytes by adjusting and maintaining intestinal balance.

[0032] The article "Microbiota-Derived Metabolites, Indole-3-aldehyde and Indole-3-acetic Acid, Differentially Modulate Innate Cytokines and Stromal Remodeling Processes Associated with Autoimmune Arthritis." used macrophages RAW 264.7 and venous endothelial cells HUVEC to study the effects of I3A on rheumatoid arthritis-related cells, but did not conduct research at the animal model and population level, and could not represent the complex pathological process of rheumatoid arthritis RA in vivo. The applicability of the experimental results in vivo may be limited; secondly, although the article explored the effects of I3A and I3AA on inflammation and bone remodeling, the discussion on their specific mechanisms of action was relatively small, especially in terms of the AhR-dependent signaling pathway that the present invention focused on, and did not clarify the mechanism. The present invention, based on animal experiments, proves the alleviating effect of I3A on rheumatoid arthritis RA from multiple perspectives, and finds that its therapeutic mechanism is mainly through the AhR-dependent signaling pathway, which is more advantageous in studying complex physiological processes and disease mechanisms in vivo. At the same time, the clear signaling pathway also improves the safety of the preparation of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The swelling of the hind paws of mice in different groups after immunotherapy, from left to right: normal group Con, model group Mod, experimental group I3A, positive control group MTX.

[0034] Figure 2 Changes in the joints of mice after immunotherapy. A: Arthritis index; B: Number of swollen joints.

[0035] Figure 3 The changes of thymus index and spleen index of mice after immunotherapy, A: thymus index; B: spleen index.

[0036] Figure 4 These are X-rays of the ankle joints of mice in different groups after immunotherapy, from left to right: normal group Con, model group Mod, experimental group I3A, and positive control group MTX.

[0037] Figure 5 The histopathological data of the ankle joints of mice in different groups after immunotherapy, A to D are: normal group Con, model group Mod, experimental group I3A, positive control group MTX; E: ankle joint histopathological score

[0038] Figure 6 is the content of inflammatory factor IL-6 in the serum of mice in different groups after immunotherapy.

[0039] Figure 7 is the content of inflammatory factor IL-1β in the serum of mice in different groups after immunotherapy.

[0040] Figure 8 is the content of inflammatory factor IL-10 in the serum of mice in different groups after immunotherapy.

[0041] Fig. 9 The expression levels of AhRmRNA and IL-10mRNA in different groups of mice after immunotherapy. DETAILED DESCRIPTION

[0042] The present invention is further described below by specific examples, but the scope of the present invention is not limited thereto. The details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall within the protection scope of the present invention.

[0043] The inventive concept of the present invention is as follows:

[0044] One hypothesis about the pathogenesis of rheumatoid arthritis is that the disease begins at the mucosal site and is the result of the interaction between the mucosal immune system and the abnormal local microbiota, which then affects the synovial joints. The drugs currently used to treat rheumatoid arthritis mainly include nonsteroidal anti-inflammatory drugs, disease-modifying antirheumatic drugs, biologics, and glucocorticoids. In addition to drug therapy, stem cell transplantation, immune purification therapy, gene therapy, and surgical therapy are all used in the treatment of RA. However, the clinical phenotype of RA patients is heterogeneous, and the treatment effects are not the same. In addition, drugs have different side effects such as kidney damage, liver toxicity, and gastrointestinal reactions. In addition to RA-related comorbidities, these increase the clinical demand for adverse prognostic factors and personalized treatment. Therefore, there is an urgent need to provide a new strategy for the treatment of rheumatoid arthritis.

[0045] Based on this, the present invention proposes an application of indole-3-carboxaldehyde in the preparation of a preparation for preventing or treating rheumatoid arthritis.

[0046] In order to enable those skilled in the art to better understand the technical solution of the present invention and implement it, the present invention is further described below in conjunction with specific embodiments and drawings. In the description of the present invention, unless otherwise specified, the reagents used are all commercially available, and the methods used are all conventional techniques in the art.

[0047] The abbreviation comparison table of the present invention is shown in Table 1.

[0048] Table 1 Abbreviations

[0049] Abbreviation Full journey I3A Indole-3-carboxaldehyde MTX Methotrexate AHr Aryl hydrocarbon receptor

[0050] Example 1

[0051] The use of I3A in the preparation of a preparation for preventing or treating rheumatoid arthritis is as follows:

[0052] (1) The intestinal microbiota metabolite I3A improves the inflammatory response in mice with collagen-induced rheumatoid arthritis.

[0053] Sixty 8-week-old Specific Pathogen Free, SPF male DBA / 1 mice were used, with a temperature of 24°C, a humidity of 60%, and a light exposure of 12 h per day, and free access to food and water. After one week of adaptive feeding, the experimental animals were randomly divided into three groups: normal group Con, model group Model, experimental group I3A, and positive control group MTX, with 10 mice in each group. The specific groups and experimental treatments are shown in Table 2.

[0054] Preparation method of modeling agent: In order to induce rheumatoid arthritis using bovine type II collagen and Freund's complete adjuvant, a complete emulsion needs to be prepared. The bovine type II collagen solution and Freund's complete adjuvant are mixed and emulsified to obtain the modeling agent.

[0055] The specific modeling method for rheumatoid arthritis is to carry out two immunizations. The first immunization is performed at multiple points in the back skin, and 100 μL of emulsifier containing 50 μg type II collagen is injected intradermally from the distal end to the proximal end. The day of the first immunization is recorded as day 0; the second immunization time is day 21, and the second immunization is still performed by vaccinating 100 μL of mixed emulsifier, which contains 50 μg type II collagen. Collagen and incomplete Freund's adjuvant are mixed and emulsified in a 1:1 ratio. The preparation method of the emulsifier is the same as the first immunization, and the needle is inserted 2 cm away from the base of the mouse tail.

[0056] After the initial immunization, the mice were scored for joint index every 2 days. The score ranged from 0 to 4:

[0057] 0 means normal; 1 means erythema and slight swelling of the ankle joint; 2 means erythema and slight swelling of the ankle joint, metatarsal joint and palmar joint; 3 means erythema and moderate swelling of the ankle joint to the metatarsophalangeal joint or palmar joint; 4 means erythema and severe swelling of the ankle joint to the toe joint. All four paws were measured, and the maximum score for each mouse was 16 points.

[0058] After the initial immunization, the redness, swelling and deformity of the mouse joints were observed, and arthritis scores were performed every 2 days. The scores for each mouse's limbs ranged from 0 to 4 points:

[0059] 0 means no swelling or redness in the joints; 1 means obvious / slight swelling or redness in the toe joints; 2 means moderate swelling or redness in the ankle joints; 3 means severe swelling or redness in the entire joint; 4 means swelling or redness in the entire paw including the ankle joints, with ankylosis. A score of 1 or above is considered an onset of disease.

[0060] Table 2 Experimental animal groups and treatment methods

[0061]

[0062] Swelling in the hind paw Figure 1 As shown, the arthritis index and the number of joint swellings are Figure 2 As shown, the thymus index and spleen index are Figure 3 As shown above, the use of I3A can relieve RA symptoms. One week after booster immunization, that is, on d27, arthritis symptoms appeared, and the inflammation peaked around d36. I3A showed that it can significantly reduce the inflammatory response during the joint onset period.

[0063] (2) The effect of intestinal flora metabolite I3A on ankle joint symptoms in mice with rheumatoid arthritis.

[0064] X-rays are commonly used to observe joints and can show bone structure and some joint diseases. For example, in the diagnosis of arthritis, X-rays can help evaluate bone changes, joint space narrowing and calcification. Figure 4 Compared with the Con group, the Mod group showed narrowing or even disappearance of the articular cartilage space, swelling of the surrounding soft tissue, increased density, bone hyperplasia, and severe deformity of the toe joints. Both I3A and MTX can effectively reduce inflammatory changes. Histopathological data show that, Figure 5 As shown in the figure, the joint space of mice in the Con group was normal, synovial cells did not proliferate, no pannus was observed, the cartilage surface was smooth and flat, chondrocytes were neatly arranged, and there was no inflammatory cell infiltration. In the Mod group, a large number of inflammatory cells infiltrated the synovium, cartilage hyperplasia caused the joint cavity to narrow significantly, chondrocyte structure was destroyed, and pannus was formed. Compared with the Mod group, I3A improved the symptoms of synovial cell hyperplasia, synovial cell erosion, pannus formation, inflammatory infiltration, and bone erosion in the joints of mice with collagen-induced arthritis CIA.

[0065] (3) The intestinal flora metabolite I3A affects the level of cytokines in the serum of rheumatoid arthritis mice.

[0066] At the end of the experiment, mice were fasted but not watered for 12 h, blood samples were collected, and after standing at room temperature for 30 min, they were centrifuged at 3000 g for 15 min at room temperature. The supernatant was taken and the content of inflammatory factors in serum was determined using a mouse ELISA kit. The mouse ELISA kit was purchased from Shanghai Jianglai Biotechnology Co., Ltd., with the IL-6 number being JL20268, the IL-1β number being JL18442, and the IL-10 number being JL20228. The operation was performed according to the instructions to determine the concentrations of inflammation-related cytokines IL-1β, IL-6, and IL-10.

[0067] The results are as follows Figure 6 to Figure 8 As shown, the mice in the model group were in a severe inflammatory state, with serum IL-1β and IL-6 levels significantly higher than those in the normal group, and IL-10 significantly lower than those in the normal group. I3A can significantly reduce the levels of IL-1β and IL-6 in serum, and significantly increase the level of IL-10. The effects of I3A and the positive control methotrexate were similar, and the results showed that I3A could affect the increase of inflammatory factors and the decrease of anti-inflammatory factors caused by arthritis.

[0068] Values ​​in the figures are means±SD.*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001vs model. #p<0.05, ##p<0.01, ###p<0.001, ####P<0.0001vs Con.

[0069] (4) The intestinal flora metabolite I3A affects the expression levels of AhR and IL-10 in the spleen of rheumatoid arthritis mice.

[0070] After the experiment, the mice were dissected according to ethical requirements, and the spleen tissue of the mice was weighed. Total RNA was extracted from the colon tissue using RNA-easy reagent for reverse transcription. The relative content of cDNA was analyzed using ChamQ SYBR qPCRMasterMix quantitative real-time PCR thermal cycler. -ΔΔCT The mRNA expression levels of AhR and IL-10 normalized by GAPDH were calculated.

[0071] Depend on Fig. 9 The quantitative results showed that the average mRNA expression of AhR in the model group was 0.06, and its average mRNA expression increased to 1.28 after I3A intervention. The average mRNA expression of IL-10 in the model group was 0.03, and the average mRNA expression of IL-10 increased significantly to 0.18 after I3A intervention.

[0072] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0073] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. Application of indole-3-carboxaldehyde in the preparation of a preparation for preventing or treating rheumatoid arthritis.

2. The use according to claim 1, characterized in that The preparation uses indole-3-carboxaldehyde as an active ingredient and is supplemented with probiotics, prebiotics and pharmaceutically acceptable excipients.

3. The use according to claim 2, characterized in that The probiotics include at least one of bifidobacterium and lactobacillus casei.

4. The use according to claim 2, characterized in that The prebiotics include at least one of fructooligosaccharides, galacto-oligosaccharides and inulin.

5. The use according to claim 2, characterized in that The pharmaceutically acceptable excipient is one or more of a diluent, a disintegrant, a precipitation inhibitor, a glidant, a binder, a dispersant, a suspending agent, an isotonic agent, a thickener, an emulsifier, a preservative and a stabilizer.

6. The use according to claim 2, characterized in that The active ingredients also include at least one of nonsteroidal anti-inflammatory drugs, glucocorticoids, sodium hyaluronate, medical chitosan and growth factors.

7. The use according to claim 6, characterized in that The non-steroidal anti-inflammatory drug includes at least one of aspirin, ibuprofen, diclofenac and celecoxib.

8. The use according to claim 6, characterized in that The glucocorticoid comprises at least one of prednisone and methylprednisolone.

9. The use according to claim 2, characterized in that The content of indole-3-carboxaldehyde in the preparation is not less than 10 mg / mL.

10. The use according to claim 1, characterized in that The acceptable dosage forms of the preparation include one of tablets, capsules, granules, injections, pills, powders, pastes and oral liquids.

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