Anti-inflammatory substance and application thereof

Abstract

The invention provides an anti-inflammatory substance and application thereof, and relates to the technical field of molecular biology. The docosahexaenoic acid can be used for effectively inhibiting expression of mRNA (messenger ribonucleic acid), protein and supernatant of IL-1beta (interleukin-1beta) of RAW264.7 cells induced by LPS (lipopolysaccharide); in addition, the docosahexaenoic acid can play a role in relieving inflammation in a transdermal administration mode, and the technical effect of inhibiting or treating inflammation in a non-invasive and safe mode is achieved.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology technology, in particular to anti-inflammatory substances and applications thereof. Background Art

[0002] Inflammatory diseases (IMIDs) are a group of common, clinically diverse conditions that include rheumatoid arthritis (RA), spondyloarthritis (SpA), connective tissue diseases, inflammatory skin diseases, inflammatory bowel disease (IBD), asthma, and autoimmune neurological diseases for which there is currently no cure.

[0003] Take rheumatoid arthritis as an example. Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease that mainly affects the joints, but may also affect other parts of the body. Common symptoms include joint swelling and pain, joint deformities, joint dysfunction, morning stiffness, etc. At present, rheumatoid arthritis cannot be cured, and the goal of treatment is to relieve symptoms and prevent disease progression. However, treatment options for rheumatoid arthritis all have certain side effects or traumatic injury defects. Therefore, how to benefit patients with rheumatoid arthritis in a non-invasive and safe way is the current focus of attention.

[0004] Docosahexaenoic Acid (DHA) is an ω-3 polyunsaturated fatty acid composed of 22 carbon atoms and 6 double bonds (C22:6). It is an important component in the human brain and retina and is essential for brain and visual development. The sources of DHA mainly include marine fish, especially cold-water fatty fish such as tuna, sardines, salmon and herring. In addition, DHA can also be synthesized in the liver through a series of enzymes by taking α-linolenic acid (ALA) as a precursor. DHA not only promotes brain development and protects vision, but is also related to cardiovascular health. It can lower triglyceride and cholesterol levels in the blood and prevent arteriosclerosis and cardiovascular and cerebrovascular diseases. Research on docosahexaenoic acid in the treatment of inflammation is still ongoing. Summary of the invention

[0005] In view of the above problems, the present invention provides anti-inflammatory compounds, especially anti-inflammatory substances and applications thereof, so as to achieve the technical effect of inhibiting or treating inflammatory diseases in a non-invasive and safe manner.

[0006] To achieve the above objectives, in a first aspect, the present invention protects an anti-inflammatory substance, the active ingredient of which is docosahexaenoic acid, which is an inhibitor of the pro-inflammatory factor IL-1β.

[0007] Furthermore, preferably, the inhibitor of the pro-inflammatory factor IL-1β is an inhibitor that inhibits the mRNA level of the pro-inflammatory factor IL-1β, or an inhibitor that inhibits the protein level of the pro-inflammatory factor IL-1β.

[0008] Furthermore, preferably, the inhibitor of the pro-inflammatory factor IL-1β is used to block LPS from activating the TLR4 / JNK / IL-1β pathway to inhibit the expression of IL-1β in serum.

[0009] Furthermore, preferably, the binding sites of the inhibitor of the pro-inflammatory factor IL-1β on the extracellular domain of the TLR4 receptor are: Y183A TLR4, S210A TLR4, E265A TLR4, S182A TLR4, R233ATLR4.

[0010] In a second aspect, the present invention protects an anti-inflammatory composition comprising the above-mentioned anti-inflammatory substance, wherein the anti-inflammatory composition is used for oral administration, injection or topical application.

[0011] In a third aspect, the present invention protects the use of an anti-inflammatory substance, which is the anti-inflammatory substance mentioned above, for treating or inhibiting inflammation or inflammation-related diseases.

[0012] Further, preferably, after interfering with the expression of TLR4 using shRNA, the anti-inflammatory function of docosahexaenoic acid disappears.

[0013] Furthermore, preferably, the forward primer sequence of the shRNA is shown as SEQ ID NO.1; and the reverse primer sequence of the shRNA is shown as SEQ ID NO.2.

[0014] Further, preferably, it is used to prepare medicines, health products, food additives, disinfectants, sanitary products, medical devices and cosmetics.

[0015] Further, preferably, the inflammation or inflammation-related diseases include sepsis, rheumatoid arthritis, systemic lupus erythematosus and related syndromes, osteoarthritis, gastrointestinal inflammation, polymyositis, dermatomyositis, vasculitis syndrome, gouty arthritis, neuroinflammation, rheumatoid arthritis, chemical pain, inflammatory pain, granuloma, granulomatous vasculitis, arteritis, skin inflammation, panniculitis, retroperitoneal fibrosis, hepatitis, pneumonia, pancreatitis, allergic inflammation, and systemic inflammatory response syndrome.

[0016] The beneficial effects of the present invention are as follows:

[0017] The anti-inflammatory compounds provided by the present invention, mainly docosahexaenoic acid, can effectively inhibit the expression of LPS-induced IL-1β in mRNA, protein and supernatant of RAW264.7 cells; and docosahexaenoic acid can alleviate the swelling of the paws of mice with rheumatoid arthritis through transdermal administration, reduce the expression of serum inflammatory factor IL-1β, reduce the infiltration of synovial inflammatory cells in the ankle joint and the degree of cartilage damage, thereby achieving the technical effect of inhibiting or treating inflammatory diseases, especially rheumatoid arthritis inflammation, in a non-invasive and safe manner. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The figure shows that docosahexaenoic acid inhibits the expression of inflammatory factors in RAW264.7 cells; A shows that docosahexaenoic acid inhibits the expression of inflammatory factor IL-1β mRNA in RAW264.7 cells; B shows that docosahexaenoic acid inhibits the expression of inflammatory factor IL-1β protein in RAW264.7 cells; C shows that docosahexaenoic acid inhibits the expression of inflammatory factor IL-1β supernatant in RAW264.7 cells;

[0019] Figure 2 The figure shows that docosahexaenoic acid inhibits the expression of inflammatory factor IL-1β through TLR4 receptor; A is the verification of the knockdown efficiency of shTLR4 in RAW264.7 cells; B is the effect of docosahexaenoic acid on the mRNA expression of LPS-induced proinflammatory factor IL-1β before and after knockdown of TLR4; C is the effect of docosahexaenoic acid on the protein expression and JNK phosphorylation of LPS-induced proinflammatory factor IL-1β before and after knockdown of TLR4;

[0020] Figure 3 Figure 1 is a diagram of docosahexaenoic acid binding to TLR4 receptor; A is a 3D diagram of docosahexaenoic acid and TLR4 binding predicted by Autoduck Vina; B is a planar diagram of docosahexaenoic acid and TLR4 binding sites predicted by Autoduck Vina; C is the five binding sites and affinities with high binding force between docosahexaenoic acid and TLR4 predicted by Autoduck Vina; D is the binding of docosahexaenoic acid and MBP protein measured by microcalorimetry; E is the binding of docosahexaenoic acid and TLR4 protein measured by microcalorimetry; F is the binding of docosahexaenoic acid and TLR4 mutant protein measured by microcalorimetry;

[0021] Figure 4The figure shows that docosahexaenoic acid alleviates the symptoms of rheumatoid arthritis in CIA mice; A is a picture of the paws of mice in the control group and the docosahexaenoic acid administration group; B is a statistical graph of the paw thickness of mice in the control group and the docosahexaenoic acid administration group; C is the expression of serum IL-1β in mice in the control group and the docosahexaenoic acid administration group; D is hematoxylin and eosin staining of ankle joint sections of mice in the control group and the docosahexaenoic acid administration group; E is safranin O fast green staining of ankle joint sections of mice in the control group and the docosahexaenoic acid administration group;

[0022] Figure 5 Figure 1 is a diagram showing the function of docosahexaenoic acid through transdermal administration; A is a fluorescence imaging diagram of skin tissue of the control group and the docosahexaenoic acid administration group labeled with anthocyanin 5; B is a frozen section of skin tissue of the control group and the docosahexaenoic acid administration group labeled with anthocyanin 5;

[0023] Figure 6 A comparison of the anti-inflammatory effects of docosahexaenoic acid and a mixture of docosahexaenoylethanolamine, ferulic acid, and naproxen. DETAILED DESCRIPTION

[0024] The present invention will be described in more detail with reference to the following examples. However, the protection scope of the present invention is not limited to the following examples.

[0025] Proinflammatory cytokines are a group of cytokines that play a key role in inflammatory responses. They can activate and regulate immune cells, promote the production of inflammatory mediators, and affect cell proliferation, differentiation, and cell death. Among them, interleukin 1β (IL-1β) is an important proinflammatory cytokine, which is mainly produced by activated macrophages and monocytes. The precursor of IL-1β is cleaved by cysteine ​​protease 1 (caspase1) in the cytoplasm to form mature IL-1β with a molecular weight of approximately 17kDa. The release of mature IL-1β requires two signals: first, the activation of Toll-like receptors (TLRs) leads to the transcription and translation of pro-IL-1β; second, NLR-induced IL-1β processing and release through a caspase 1-dependent mechanism. The detection and analysis of IL-1β is an important aspect of studying inflammation and immune responses.

[0026] Docosahexaenoic Acid (DHA) is a polyunsaturated fatty acid essential to the human body. DHA (CAS No. 6217-54-5 and 25167-62-8) is a straight-chain fatty acid containing 22 carbon atoms and six unsaturated double bonds. DHA exists in two main forms: triglyceride form: This is the common form of DHA in fish oil, usually obtained from deep-sea fish oil rich in DHA and EPA through molecular distillation. This form of DHA contains a mixture of Omega-3 fatty acids.

[0027] . Ethyl ester form: DHA can also exist in the form of ethyl ester, which is more easily absorbed by the human body and is often used in dietary supplements and certain foods for special medical purposes.

[0028] Example 1

[0029] 1. Materials and Methods

[0030] 1.1 Experimental Materials

[0031] 1.1.1 Cell lines: Mouse monocyte-macrophage cell line RAW264.7 and human embryonic kidney cell line (293FT) were purchased from the Cell Bank of the Chinese Academy of Sciences / Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

[0032] 1.1.2 Mice: Male DBA / 1 mice were purchased from Vital River.

[0033] 1.1.3 Experimental drugs and reagents: DMEM high-glucose medium, phosphate-buffered saline (PBS), fetal bovine serum (FBS): Gibco; dimethyl sulfoxide (DMSO), trypsin, lipopolysaccharide (LPS): Sigma; complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA), bovine type II collagen: Chondrex; IL-1β ELISA kit: Huamei Biotechnology; TRIzol: Thermo Fisher; reverse transcription kit, quantitative enzyme: Novazon Biotechnology Co., Ltd.; IL-1β primary antibody: R&D Company; HSP90, p-JNK, JNK primary antibody: CST Company; RIPA cell lysate: Beyotime; Docosahexaenoic acid (C22:6): Shanghai Lanmu Chemical Technology Co., Ltd.; Vitamin E emulsion, Guangzhou Biaoting Pharmaceutical Biotechnology Co., Ltd.

[0034] 1.2 Experimental methods and results

[0035] 1.2.1 Docosahexaenoic acid inhibits the expression of pro-inflammatory factors in RAW264.7 cells

[0036] Lipopolysaccharide (LPS) is the main component of the cell wall of Gram-negative bacteria. It is antigenic and is released when Gram-negative bacteria break apart. It is a strong pyrogen. In animal and cell experiments, LPS is widely used to induce inflammation. Generally, macrophages RAW264.7 are used as research objects, and LPS is used to induce macrophages to produce inflammatory factors and promote the expression of genes related to inflammation to create an inflammation model.

[0037] RAW264.7 macrophages in the logarithmic growth phase were seeded in 12-well culture plates at a cell density of 1×10 5 / mL, 1mL per well, cultured overnight at 37°C, 5% CO2. When the cell density reached about 80%, DMSO and C22:6 were added to the control group and the experimental group, respectively, for 12 hours. LPS (final concentration of 1μg / mL) was added to each well and incubated for 12 hours. Each group was treated with 6 wells. After the treatment, the cell supernatant was collected, and the expression of IL-1β in the supernatant was detected by ELISA kit, the cell RNA was extracted by Trizol, and the cell protein was extracted by RIPA lysis buffer for IL-1β detection.

[0038] result

[0039] Docosahexaenoic acid significantly inhibited the expression of IL-1β mRNA in RAW264.7 cells induced by LPS. Figure 1 -A; compared with the control group, docosahexaenoic acid reduced the expression of LPS-induced IL-1β by about 1 / 2.

[0040] Docosahexaenoic acid significantly inhibited the expression of IL-1β protein in RAW264.7 cells induced by LPS. Figure 1 -B shows that docosahexaenoic acid significantly reduced the expression of pro- and mature IL-1β induced by LPS.

[0041] Docosahexaenoic acid significantly inhibited the expression of IL-1β in the supernatant of RAW264.7 cells induced by LPS. Figure 1 As shown in Figure 5C, docosahexaenoic acid reduced the amount of IL-1β secreted into the cell supernatant induced by LPS by 1 / 3 compared with the control group.

[0042] In summary, the docosahexaenoic acid provided by the present invention can effectively inhibit the expression of IL-1β in mRNA, protein and supernatant of RAW264.7 cells induced by LPS.

[0043] 1.2.2 Docosahexaenoic acid inhibits the expression of pro-inflammatory factor IL-1β through TLR4

[0044] TLR4 receptor is an important receptor in the inflammatory pathway, so shRNA-TLR4 was constructed and synthesized. Using the lentiviral packaging system, shTLR4 and auxiliary plasmids PMD2G and PSPAX were transfected into 293FT cells, and the virus was collected after 48 hours. The virus was used to infect RAW264.7 cells to obtain a cell line with stable knockdown of TLR4. The effect of docosahexaenoic acid on the expression of inflammatory factors in TLR4 knockdown cells was explored according to the method in 1.2.2.

[0045] result

[0046] The forward primer sequence of shRNA-TLR4 is shown in SEQ ID NO.1, and the reverse primer sequence is shown in SEQ ID NO.2. Figure 2 -A, the knockdown efficiency is about 50%.

[0047] SEQ ID NO.1: 5'-CCGGCCGTTGGTGTATCTTTGAATACTCG

[0048] AGTATTCAAAGATACACCAACGGTTTTG-3'

[0049] SEQ ID NO.2:5'-AATTCAAAAACCGTTGGTGTATCTTTGAA

[0050] TACTCGAGTATTCAAAGATACACCAACGG-3'

[0051] After knocking down TLR4, docosahexaenoic acid could not significantly inhibit the expression of LPS-induced proinflammatory factor IL-1β mRNA in RAW264.7 cells. Figure 2 As shown in (B), compared with the PLKO.1 group, docosahexaenoic acid in the shTLR4 group could not significantly reduce LPS-induced IL-1β mRNA expression.

[0052] After knocking down TLR4, docosahexaenoic acid could not significantly inhibit the protein expression of pro-inflammatory factor IL-1β in RAW264.7 cells induced by LPS. Figure 2 -B, compared with the PLKO.1 group, docosahexaenoic acid in the shTLR4 group could not significantly reduce the protein expression levels of LPS-induced IL-1β and phosphorylated JNK.

[0053] 1.2.3 Docosahexaenoic acid binds to TLR4 receptor

[0054] In order to verify the mode of action of docosahexaenoic acid and TLR4, the binding sites of TLR4 extracellular domain and docosahexaenoic acid were predicted using Autodock Vina software. In order to verify the prediction results, the TLR4 extracellular domain and mutant sequences were inserted into the pmal-C5X vector, and the TLR4 and TLR4 mutant proteins were purified using the E. coli system. In order to exclude the influence of MBP on the pmal-C5X vector, MBP protein was also purified separately. The dissociation constants of docosahexaenoic acid with MBP, TLR4 and TLR4 mutants were detected using a microcalorimeter to determine whether there was a possibility of binding.

[0055] result

[0056] Autoduck Vina software predicted that there are potential binding sites for docosahexaenoic acid on the extracellular domain of TLR4, such as Figure 3 -A shows the three-dimensional binding mode between TLR4 and docosahexaenoic acid. Figure 3 -B is the potential binding site for the two. Figure 3 -C represents five binding sites with higher binding force. The binding sites of docosahexaenoic acid on the extracellular domain of TLR4 receptor include Y183A TLR4, S210A TLR4, E265A TLR4, S182A TLR4 and R233A TLR4.

[0057] The potential binding capacity of docosahexaenoic acid and three proteins was measured by microcalorimetry. Figure 3 -D shows that docosahexaenoic acid and MBP protein do not bind; Figure 3 -E, docosahexaenoic acid binds to TLR4 with a dissociation constant of 1.53 μ m ; Figure 3 -F, Docosahexaenoic acid and TLR4 mutants do not bind.

[0058] 1.2.4 Docosahexaenoic acid alleviates rheumatoid arthritis symptoms in CIA mice

[0059] Docosahexaenoic acid and vitamin E emulsion were fully mixed and evenly applied to the paws of mice with collagen-induced rheumatoid arthritis (CIA). The control group was only applied with vitamin E emulsion once a day for 3 consecutive weeks. The thickness of the mouse paws was measured and recorded with a vernier caliper every week. After 3 weeks, the mouse serum was taken to detect the expression of IL-1β using an ELISA kit. The ankle joint tissue of the mouse was fixed with 4% paraformaldehyde for 48 hours and then decalcified for 1-2 months. After paraffin embedding, the sections were sliced ​​and stained with hematoxylin and eosin to determine the infiltration of inflammatory cells, and stained with safranin O fast green to determine the cartilage damage.

[0060] result

[0061] Compared with the control group, the swelling of the paws of mice in the docosahexaenoic acid group was significantly reduced. Figure 4 -A and 4-B, the swelling of the paws of mice in the docosahexaenoic acid group was lower, and the average paw thickness was about 0.7 mm lower than that in the control group.

[0062] The expression levels of serum inflammatory factors were detected and it was found that the expression of serum IL-1β and IL-2 in the docosahexaenoic acid group was significantly lower than that in the control group. Figure 4 -C shows that the expression level of serum IL-1β in the docosahexaenoic acid group of mice was reduced by nearly 1 / 2 compared with that in the control group.

[0063] Pathological sections of ankle joints showed that the infiltration of inflammatory cells in the ankle synovium of mice in the docosahexaenoic acid group was reduced compared with that in the control group. Figure 4 -D shows that the number of inflammatory cells pointed by the yellow arrows decreased; the degree of cartilage damage was significantly reduced. Figure 4 -The cartilage integrity is better at the position of the red arrow shown in E.

[0064] In a specific implementation process, docosahexaenoic acid is used as an external medicine or cosmetic for treating rheumatoid arthritis. The dosage form of the external medicine can be creams: semi-solid dosage forms, containing drugs and emulsifiers, suitable for the skin surface. Among them, the emulsifier can be vitamin E. It can also be gels, lotions, sprays, foams, patches, solutions, plasters, etc. It is not specifically limited here.

[0065] 1.2.5 Docosahexaenoic acid works through transdermal administration

[0066] To prove that docosahexaenoic acid can work through transdermal administration, CY5 dye was labeled on the right carboxyl group of docosahexaenoic acid. Docosahexaenoic acid-CY5 was fully mixed with vitamin E emulsion and applied to the surface of mouse skin, shielded from light. The fluorescence of the outer and inner surfaces of the skin was counted using a small animal live imaging device, and the skin tissue was cut into 20μm slices using a frozen microtome, and the location of fluorescence leakage was observed using a confocal microscope.

[0067] result

[0068] The imaging device showed that docosahexaenoic acid-CY5 can penetrate the skin tissue, such as Figure 5-A shows that compared with the control group, both the outer epidermis (ROI=1.811e+12) and inner epidermis (ROI=1.436e+12) of the skin of mice in the docosahexaenoic acid-CY5 group showed fluorescence, and the fluorescence intensity of the outer epidermis was 3.75e+11 greater than that of the inner epidermis.

[0069] Skin tissue frozen sections show that the dermis of the skin tissue has red fluorescence. Figure 5 -B shows that the red fluorescence of the docosahexaenoic acid group leaks into the dermis of the skin, indicating that docosahexaenoic acid-CY5 can reach the dermis of the skin tissue through transdermal absorption and thus play a role in alleviating rheumatoid arthritis.

[0070] In a specific implementation process, the inflammation or inflammation-related diseases to which the present invention is applicable may include sepsis, rheumatoid arthritis, systemic lupus erythematosus and related syndromes, osteoarthritis, gastrointestinal inflammation, polymyositis, dermatomyositis, vasculitis syndrome, gouty arthritis, neuroinflammation, rheumatoid arthritis, chemical pain, inflammatory pain, granuloma, granulomatous vasculitis, arteritis, skin inflammation, panniculitis, retroperitoneal fibrosis, hepatitis, pneumonia, pancreatitis, allergic inflammation, and systemic inflammatory response syndrome.

[0071] In a specific implementation, the cosmetically or pharmaceutically effective amount of the docosahexaenoic acid of the present invention to be administered for treating a disorder, disease and / or condition and the dosage thereof depend on various factors, including the age, severity of the disorder, disease or condition of the patient, the route and frequency of administration and on the specific nature of the docosahexaenoic acid to be used. A "cosmetically or pharmaceutically effective amount" refers to a non-toxic amount of docosahexaenoic acid sufficient to provide the desired effect. The docosahexaenoic acid of the present invention used in the cosmetic composition or pharmaceutical composition of the present invention is a concentration that is cosmetically or pharmaceutically effective to achieve the desired effect.

[0072] Formulations for topical or transdermal application (topical application) may be in any solid, liquid or semisolid dosage form, such as, for example and without limitation, creams, composite emulsions such as, for example and without limitation, emulsions of oil and / or silicone in water, emulsions of water in oil and / or silicone, emulsions of the water / oil / water or water / silicone / water type, and emulsions of the oil / water / oil or silicone / water / silicone type, anhydrous compositions, aqueous dispersions, oils, emulsions, balms, foams, lotions, gels, cream gels, hydroalcoholic solutions, hydroglycolic solutions, liniments, saline solutions, soaps, shampoos, conditioners, serums, polysaccharide films, ointments, mousses, saline solutions, powders, sticks, pencils and aerosols or sprays, including leave-on formulations and rinse-off formulations. Or they can be added to different cosmetic product lines, such as makeup foundations, such as, for example, liquid or solid makeup foundations, makeup removal washes, makeup removal emulsions, under eye concealers, eye shadows, lipsticks, lip protectors, lip glosses and powders, and others.

[0073] The docosahexaenoic acid of the present invention can also be added to disinfectants, sanitary products or medical devices that directly contact the skin, mucous membranes and / or scalp to treat and / or inhibit inflammation or inflammation-related diseases caused by overexpression of the proinflammatory factor IL-1β. Preferably, the medical device is a bandage, gauze, diaper, sanitary napkin, dressing, bed cover, wipe, hydrogel, adhesive patch, non-adhesive patch, microcurrent patch and / or mask.

[0074] Comparative Example 1

[0075] In order to emphasize the anti-inflammatory effect of docosahexaenoic acid, docosahexaenoic acid, a mixture of docosahexaenoylethanolamine (DHEA) and ferulic acid, and a mixture of docosahexaenoylethanolamine and naproxen were simultaneously treated with RAW264.7 cells with a density of 80% for 12 hours; it should be noted that, as far as those skilled in the art know, the anti-inflammatory effect of the mixture of docosahexaenoylethanolamine (DHEA) and ferulic acid and the mixture of docosahexaenoylethanolamine and naproxen is equivalent to the anti-inflammatory effect of the derivatives of docosahexaenoic acid.

[0076] Then the cells were treated with LPS for 12 hours. Cell RNA was extracted with Trizol to detect the expression of IL-1β mRNA level.

[0077] result

[0078] It was found that the inhibitory effect of docosahexaenoic acid on IL-1β was superior to that of the mixture of the two drugs. Figure 6 As shown, docosahexaenoic acid can reduce the expression of LPS-induced IL-1β by about 1 / 2, but the other two groups of drugs (a mixture of docosahexaenoylethanolamine (DHEA) and ferulic acid and a mixture of docosahexaenoylethanolamine and naproxen) only have a weak inhibitory effect on IL-1β.

[0079] In summary, the docosahexaenoic acid provided by the present invention can effectively inhibit the expression of IL-1β in the mRNA, protein and supernatant of RAW264.7 cells induced by LPS; and docosahexaenoic acid can inhibit rheumatoid arthritis by transdermal administration, achieving the technical effect of inhibiting or treating rheumatoid arthritis in a non-invasive and safe manner. The anti-inflammatory substance with docosahexaenoic acid as the active ingredient of the present invention has an effective effect of inhibiting or treating inflammation, which provides a guarantee for its wide application in the fields of medicines, health products, food additives, disinfectants, sanitary products, medical devices and cosmetics.

[0080] Among the various experimental supplies mentioned in this article (including but not limited to: chemical reagents, biological products, cells, organisms, instruments, etc.), for those that are special or difficult to obtain, the manufacturers, references or detailed preparation methods have been indicated in the article; those not otherwise specified are conventional experimental supplies and can be easily obtained by various means (such as purchase, self-preparation, etc.) before the date of this application.

[0081] Although the present invention has been described in detail above by general description, specific implementation methods and experiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications and improvements made without departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Claims

1. An anti-inflammatory substance, characterized in that The active ingredient of the anti-inflammatory substance is docosahexaenoic acid, which is an inhibitor of the pro-inflammatory factor IL-1β.

2. The anti-inflammatory substance according to claim 1, characterized in that The inhibitor of the pro-inflammatory factor IL-1β is an inhibitor that inhibits the mRNA level of the pro-inflammatory factor IL-1β, or an inhibitor at the protein level.

3. The anti-inflammatory substance according to claim 2, characterized in that The inhibitor of the pro-inflammatory factor IL-1β is used to block LPS from activating the TLR4 / JNK / IL-1β pathway to inhibit the expression of IL-1β in serum.

4. The anti-inflammatory substance according to claim 1, characterized in that The binding sites of the inhibitor of the pro-inflammatory factor IL-1β on the extracellular domain of the TLR4 receptor are: Y183A TLR4, S210A TLR4, E265A TLR4, S182A TLR4, and R233ATLR4.

5. An anti-inflammatory composition, characterized in that The anti-inflammatory composition comprises the anti-inflammatory substance according to claim 1, and is used for oral administration, injection or topical application.

6. Use of an anti-inflammatory substance, wherein the anti-inflammatory substance is the anti-inflammatory substance according to claim 1, characterized in that: Used to treat or inhibit inflammation or inflammation-related diseases.

7. The use according to claim 6, characterized in that: After interfering with TLR4 expression using shRNA, the anti-inflammatory function of docosahexaenoic acid disappeared.

8. The use according to claim 7, characterized in that: The forward primer sequence of the shRNA is shown in SEQ ID NO.1; the reverse primer sequence of the shRNA is shown in SEQ ID NO.

2.

9. The use according to claim 6, characterized in that: Used in the preparation of medicines, health products, food additives, disinfectants, sanitary products, medical devices and cosmetics.

10. The use according to claim 6, characterized in that: The inflammation or inflammation-related diseases include sepsis, rheumatoid arthritis, systemic lupus erythematosus and related syndromes, osteoarthritis, gastrointestinal inflammation, polymyositis, dermatomyositis, vasculitis syndrome, gouty arthritis, neuroinflammation, rheumatoid arthritis, chemical pain, inflammatory pain, granuloma, granulomatous vasculitis, arteritis, skin inflammation, panniculitis, retroperitoneal fibrosis, hepatitis, pneumonia, pancreatitis, allergic inflammation, and systemic inflammatory response syndrome.

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