Application of sophoranol A10 in preparation of anti-inflammatory products

Abstract

The application provides application of sophoranol A10 or a pharmaceutically acceptable salt thereof in preparation of an anti-inflammatory product, and a method for inhibiting activation of macrophages in vitro for non-treatment purposes. The application firstly proves that sophoranol A10 has high protein affinity with S100A8 / A9, and can inhibit production of cell inflammatory factors, inhibit activation of macrophages and regulate inflammatory response of the body through a TLR4 / NFκB / MyD88 signal pathway. Therefore, sophoranol A10 can be used for preparation of an anti-inflammatory product, and has a good market application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of euchrenone A10 in preparation of anti-inflammatory products. BACKGROUND

[0002] Euchrenone A10 is mainly extracted from leguminous plants, chemically synthesized and biologically engineered, and has a molecular formula of C 25 H 26 O5, a molecular weight of 406.5 and a structural formula as shown in the following:

[0003]

[0004] Inflammation, also known as “inflammation” in common parlance, is a defense response of the body to stimulation, and is manifested as redness, swelling, heat, pain and dysfunction. Inflammation can be infectious inflammation caused by infection, or non-infectious inflammation not caused by infection. Macrophages are a kind of white blood cells located in tissues and derived from monocytes. Macrophages, as the creators of the tissue microenvironment, are widely involved in and run through the pathological process of inflammation, and can release a variety of inflammatory mediators to cause corresponding inflammatory pathways to be activated. Therefore, by regulating the activation of macrophages, excessive inflammatory response can be inhibited, thereby playing an anti-inflammatory role and preventing and treating diseases related to excessive inflammatory response.

[0005] S100A8 and S100A9 are main members of the S100 protein family of the S100A8 / S100A9 multifunctional calcium-binding protein family, and exist in the form of a dimer S100A8 / A9. They are mainly derived from activated neutrophils, monocytes, macrophages and dendritic cells, and are involved in the occurrence and development of various inflammatory diseases. SUMMARY

[0006] Therefore, the purpose of the present application is to provide application of euchrenone A10 in preparation of anti-inflammatory products. Euchrenone A10 can inhibit the activation of macrophages, reduce the expression of IL-6 and inhibit the expression of TLR4 / NFκB / MyD88 signal pathway-related genes.

[0007] To achieve the above purpose, the technical scheme is as follows.

[0008] Euchrenone A10 or a pharmaceutically acceptable salt thereof is applied in preparation of anti-inflammatory products.

[0009] In some embodiments, the anti-inflammatory product can inhibit the activation of macrophages.

[0010] In some embodiments, the anti-inflammatory product can inhibit the expression of S100A8 genes and / or S100A9 genes.

[0011] In some embodiments, the anti-inflammatory product can inhibit the expression of IL-6.

[0012] In some embodiments, the anti-inflammatory product can inhibit the TLR4 / NFκB / MyD88 signaling pathway.

[0013] The present application also provides an anti-inflammatory drug comprising sophoranone A10 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

[0014] In some embodiments, the dosage form of the drug includes granules, tablets, solutions, pills, tinctures, powders, injections, extracts, ointments, capsules.

[0015] The present application also provides a method for inhibiting the activation of macrophages in vitro for non-therapeutic purposes, comprising the steps of: adding sophoranone A10 or a pharmaceutically acceptable salt thereof to a macrophage in vitro culture system for treatment, and then adding a macrophage activation inducer; the final concentration of the sophoranone A10 or the pharmaceutically acceptable salt thereof in the culture system is 0.1-1000 μM.

[0016] In some embodiments, the final concentration of the sophoranone A10 or the pharmaceutically acceptable salt thereof in the culture system is 1-500 μM; preferably, the final concentration of the sophoranone A10 or the pharmaceutically acceptable salt thereof in the culture system is 5-200 μM; more preferably, the final concentration of the sophoranone A10 or the pharmaceutically acceptable salt thereof in the culture system is 10-150 μM.

[0017] In some embodiments, the macrophage activation inducer is selected from at least one of lipopolysaccharide LPS and calprotectin S100A8 / A9.

[0018] The present application has first confirmed that sophoranone A10 has high protein affinity with S100A8 / A9, and can inhibit the production of cell inflammatory factors and the activation of macrophages through the TLR4 / NFκB / MyD88 signaling pathway, and regulate the inflammatory response of the body. Therefore, sophoranone A10 can be used for preparing anti-inflammatory products, and has good market application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 Molecular docking diagram of sophoranone A10 and calcium binding protein S100A8 / A9 interaction.

[0020] Figure 2 Results of bio-layer interferometry (BLI) for detecting the protein affinity of sophoranone A10 and calcium binding protein S100A8 / A9.

[0021] Figure 3 The results of the influence of sophoranone A10 on the expression of S100A8, S100A9 and IL-6 mRNA in RAW264.7 macrophages.

[0022] Figure 4 The results of the mechanism of the influence of sophoranone A10 on S100A8 / A9 in RAW264.7 macrophages.

[0023] Figure 5 The results of the mechanism of the influence of sophoranone A10 on the stimulation of RAW264.7 macrophages by exogenous S100A8 / A9 protein. DETAILED DESCRIPTION

[0024] The experimental methods in the following examples of the present application, unless otherwise specified, are generally carried out according to the conventional conditions, or according to the conditions suggested by the manufacturers. The various common chemical reagents used in the examples are all commercially available products.

[0025] Unless otherwise defined, all technical and scientific terms used in the present application have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0026] The terms "comprising" and "having" and any variations thereof used in the present application are intended to cover a non-exclusive inclusion. For example, a process, method, device, product or apparatus that comprises a list of steps does not necessarily limit the process, method, device, product or apparatus to only those steps listed, but can optionally include additional steps not listed, or optionally include other steps inherent to such process, method, product or apparatus.

[0027] In the present application, "a plurality of" refers to two or more. "And / or", which describes the association relationship of the associated objects, means that there can be three relationships, for example, A and / or B can mean that there are three cases of A alone, A and B together, and B alone. The character " / " generally represents that the associated objects before and after are in an "or" relationship.

[0028] The following will be described in conjunction with specific examples.

[0029] Example 1 Interaction between sophoranone A10 and calcium-binding protein S100A8 / A9

[0030] The interaction between Euchrenone A10 and S100A8 / A9 was determined by searching for protein active ligands in a workstation by molecular docking calculation method after checking the three-dimensional structure of Euchrenone A10 and S100A8 / A9 in the corresponding database. The results showed that Euchrenone A10 had interaction with S100A8 / A9 (Table 1, Figure 1 ).

[0031] Table 1 Computer molecular simulation docking score of interaction between Euchrenone A10 and S100A8 / A9

[0032]

[0033] Example 2 Affinity test of Euchrenone A10 and S100A8 / A9

[0034] After Euchrenone A10 was diluted by a two-fold gradient dilution method to 6 concentration gradients, it was detected with the prepared S100A8 / A9 probe on a molecular interaction instrument. The detection results showed that Euchrenone A10 had strong affinity with S100A8 / A9 protein (Kd = 34.7 ± 9.14). Figure 2

[0035] Example 3 Effect of Euchrenone A10 on the expression of S100A8, S100A9 and IL-6 mRNA in RAW264.7 macrophages

[0036] 1×10 5 RAW264.7 cells were inoculated in a 6-well plate and cultured for 24 h, and then the following groups were set up: (1) Control group: no treatment; (2) Model group: after 2 h of continuous culture, LPS was added to stimulate induction at a final concentration of 100 ng / ml; (3) Paquinimod group: after RAW264.7 cells were treated with paquinimod at a final concentration of 5 μM for 2 h, LPS was added to stimulate induction at a final concentration of 100 ng / ml; (4) Euchrenone A10 group: after RAW264.7 cells were treated with Euchrenone A10 diluted with DMEM medium to different concentrations (20 μM, 40 μM, 80 μM) for 2 h, LPS was added to stimulate induction at a final concentration of 100 ng / ml. After 24 h of LPS stimulation and induction, the cells in each group were collected, and the expression levels of S100A8, S100A9 and IL-6 were detected by PCR. The results showed that 20-80 μM of Euchrenone A10 could significantly inhibit the expression levels of S100A8, S100A9 and IL-6 mRNA in LPS-induced RAW264.7 cells, and had a dose-dependent relationship ( Figure 3 ).​

[0037] Example 4 Mechanism of Euchrenone A10 on the effect of S100A8 / A9 in RAW264.7 macrophages

[0038] 1 x 10 5 RAW264.7 cells were seeded in 6-well plates for 24 h, and then divided into the following groups: (1) Control group: no treatment; (2) Model group: after 2 h of continuous culture, RAW264.7 cells were stimulated by LPS at a final concentration of 100 ng / ml; (3) Paquinimod group: RAW264.7 cells were treated with paquinimod at a final concentration of 5 μM for 2 h, and then stimulated by LPS at a final concentration of 100 ng / ml; (4) Euchrenone A10 group: RAW264.7 cells were treated with Euchrenone A10 at different concentrations (20 μM, 40 μM, 80 μM) for 2 h, and then stimulated by LPS at a final concentration of 100 ng / ml. After 24 h of LPS stimulation, cells in each group were collected, and the expression levels of TLR4, NFKBp65, MyD88, TRAF6, RAGE, IRAK were detected by PCR. The results showed that Euchrenone A10 at a concentration of 20-80 μM could significantly inhibit the expression levels of TLR4, NFKBp65, MyD88, TRAF6, RAGE, IRAK mRNA in LPS-induced RAW264.7 cells, and had a dose-dependent relationship. Therefore, the mechanism of Euchrenone A10 on the effect of S100A8 / A9 in RAW264.7 macrophages is related to the TLR4 / NFκBp65 / MyD88 signaling pathway. Figure 4

[0039] Example 5 Mechanism of Euchrenone A10 on the effect of exogenous S100A8 / A9 protein on RAW264.7 macrophages

[0040] 1 x 10 5 ​RAW264.7 cells were inoculated in 6-well plates and cultured for 24 h, and then divided into the following groups: (1) Control group: no treatment; (2) Model group: after being cultured for 2 h, RAW264.7 cells were stimulated and induced by adding S100A8 / A9 protein at a final concentration of 2 μg / ml; (3) Paquinimod group: after RAW264.7 cells were treated with paquinimod at a final concentration of 5 μM for 2 h, RAW264.7 cells were stimulated and induced by adding S100A8 / A9 protein at a final concentration of 2 μg / ml; (4) Euchrenone A10 group: after RAW264.7 cells were treated with Euchrenone A10 at different concentrations (20 μM, 40 μM, 80 μM) for 2 h, RAW264.7 cells were stimulated and induced by adding S100A8 / A9 protein at a final concentration of 2 μg / ml. After being stimulated and induced by S100A8 / A9 protein for 24 h, cells in each group were collected, and the expression levels of IL-6, TLR4, NFKBp65, MyD88, TRAF6, RAGE and IRAK were detected by PCR. It was found that Euchrenone A10 at 20-80 μM can significantly inhibit the mRNA expression levels of IL-6, TLR4, NFKBp65, MyD88, TRAF6, RAGE and IRAK in RAW264.7 cells induced by S100A8 / A9 protein, and the inhibition has a dose-dependent relationship. Therefore, the mechanism of action of Euchrenone A10 on RAW264.7 macrophages stimulated by exogenous S100A8 / A9 protein is related to the TLR4 / NFKBp65 / MyD88 signaling pathway. Figure 5

[0041] In summary, Euchrenone A10 can inhibit the activation of macrophages and inhibit the secretion of inflammatory factors, and can be used for preparing anti-inflammatory products.

[0042] Each technical feature of the above-described embodiments can be combined arbitrarily, and to make the description concise, each technical feature in the above embodiments is not described in all possible combinations, however, as long as the combination of technical features does not exist contradictory, it should be considered as the scope of the present disclosure.

[0043] The above-described embodiments only express several implementation manners of the present application, and the description is more specific and detailed, but it should not be understood as a limitation on the scope of the patent of the present application. It should be pointed out that for ordinary skilled persons in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.​

Claims

1. The use of Sophora flavescens A10 or a pharmaceutically acceptable salt thereof as the sole active ingredient in the preparation of anti-inflammatory products, characterized in that, The anti-inflammatory product can inhibit macrophage activation, inhibit the expression of S100A8 and / or S100A9 genes, inhibit IL-6 expression, and inhibit the TLR4 / NFκB / MyD88 signaling pathway.

2. The use of sophora flavescens A10 or a pharmaceutically acceptable salt thereof as the sole active ingredient in the preparation of an anti-inflammatory product, as described in claim 1, is characterized in that... The anti-inflammatory product contains sophorone A10 or a pharmaceutically acceptable salt thereof, as well as pharmaceutically acceptable excipients.

3. The use of Sophora flavescens A10 or a pharmaceutically acceptable salt thereof as the sole active ingredient in the preparation of an anti-inflammatory product, as described in claim 1, is characterized in that... The anti-inflammatory products include anti-inflammatory drugs, and the dosage forms of the anti-inflammatory drugs include granules, tablets, solutions, pills, tinctures, powders, injections, extracts, ointments, and capsules.

4. A method for inhibiting macrophage activation in vitro for non-therapeutic purposes, characterized in that, The method includes the following steps: adding Sophora tonkinensis A10 or a pharmaceutically acceptable salt thereof to a macrophage in vitro culture system for treatment, and then adding a macrophage activation inducer; the final concentration of Sophora tonkinensis A10 or a pharmaceutically acceptable salt thereof in the culture system is 5-200 μM.

5. The method as described in claim 4, characterized in that, The macrophage activation inducer is selected from at least one of lipopolysaccharide (LPS) and calprotectin S100A8 / A9.

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