Application of diaryl heptane compound in preparation of antioxidant and / or anti-inflammatory products

Abstract

The invention discloses an application of a diaryl heptane compound in preparation of an antioxidant and / or anti-inflammatory product. The structural formula of the diaryl heptane compound (DH) is as follows: # imgabs0. The compound DH can regulate and control release of reactive oxygen species (ROS) by inhibiting mitochondrial membrane potential, and can activate an Nrf2 / HO-1 signal channel, inhibit expression of NF-kB in nucleus, inhibit activation of an MAPK signal channel and expression of iNOS and COX-2. Besides, DH also has the effect of activating a PI3K / Akt / mTOR signal channel to generate negative feedback, is beneficial to further enhancing the inhibition effect on an MAPK channel and NF-kB, and can be widely applied to preparation of antioxidant and / or anti-inflammatory drugs.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of a diarylheptane compound in the preparation of an antioxidant and / or anti-inflammatory product. Background Art

[0002] Oxidative stress refers to the excessive production of reactive oxygen species (ROS) in cells and tissues, which cannot be neutralized by the antioxidant system. Oxidative stress can activate a variety of transcription factors, leading to differences in the expression of some genes involved in inflammatory pathways. Nrf2 is the main regulator of the antioxidant defense system in the body and participates in signal transduction related to a variety of intracellular defense mechanisms. Under oxidative stress, Nrf2 will be activated and enter the cell nucleus, bind to the antioxidant response element, and show obvious antioxidant effects. The Nrf2 / HO-1 pathway plays an important regulatory role in the occurrence and regulation of oxidative stress response.

[0003] Lipopolysaccharide (LPS) is a macromolecular complex present in the outer membrane of Gram-negative bacteria. LPS is one of the main components of bacteria and has a strong immune activation effect. LPS has a very close relationship with macrophages and is often used as an inducer of oxidative stress response in research. When LPS binds to receptors on the surface of macrophages (such as Toll-like receptor 4, TLR4), it can activate a series of signaling pathways, including NF-κB, MAPK, etc., promote the release of reactive oxygen species ROS and proinflammatory cytokines (such as IL-1β and IL-6), and then induce oxidative stress response. In the related art, diarylheptane compounds are a general term for a class of compounds formed by two aromatic rings oxidized or substituted to different degrees and connected at the 1,7-position by an oxidized fatty chain alkane with 7 carbon atoms. This class of compounds is mainly found in the roots, stems, skins, flowers and peels of plants. Due to its unique chemical structure and wide range of pharmacological activities, it has attracted widespread attention from researchers at home and abroad. However, there are few studies and reports on its antioxidant activity.

[0004] Based on this, there is an urgent need to seek a diarylheptane compound with excellent antioxidant activity and its application in the preparation of antioxidant and / or anti-inflammatory products. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes an application of a diarylheptane compound in the preparation of an antioxidant and / or anti-inflammatory product. The present invention finds that the diarylheptane compound DH can inhibit the decrease of mitochondrial membrane potential and the generation of ROS in macrophages, inhibit oxidative stress, inhibit the release of inflammatory factors NO, IL-6 and IL-1β, inhibit the expression of iNOS and COX-2 proteins and genes, inhibit the expression of inflammatory factors IL-6, IL-1β, MCP-1, CCL2 and IFN-γ genes, inhibit the activation of MAPK pathway and NF-κB pathway, activate Nrf2 / HO-1 pathway, activate PI3K pathway to produce negative regulation, thereby inhibiting the development of oxidative stress response and inflammation.

[0006] In a first aspect of the present invention, there is provided a use of a diarylheptane compound as shown in Formula I or a pharmaceutically acceptable salt, ester, solvate, or prodrug thereof in the preparation of an antioxidant and / or anti-inflammatory product, wherein:

[0007]

[0008] The application of the embodiments of the present invention has at least the following beneficial effects:

[0009] The present invention has experimentally proved that the diarylheptane compound DH of the present invention can achieve its antioxidant and anti-inflammatory effects by inhibiting the mitochondrial membrane potential to regulate the release of reactive oxygen species ROS and activate the Nrf2 / HO-1 signaling pathway. Further studies have found that the compound DH can inhibit the expression of NF-κB in the nucleus, inhibit the activation of the MAPK signaling pathway, and inhibit the expression of iNOS and COX-2. In this process, DH also has a negative feedback effect of activating the PI3K / Akt / mTOR signaling pathway, which helps to further enhance the inhibitory effect on the MAPK pathway and NF-κB.

[0010] In addition, the compound DH of the present invention is safe and effective to cells within a concentration range of 0 to 100 μM, has no toxic side effects, and can be used to prepare antioxidants and / or anti-inflammatory drugs to eliminate oxidative stress and inflammatory reactions.

[0011] The second aspect of the present invention provides the use of a diarylheptane compound as shown in Formula I or a pharmaceutically acceptable salt, ester, solvate, or prodrug thereof in the preparation of a mitochondrial membrane potential inhibitor and / or a reactive oxygen species inhibitor.

[0012] The third aspect of the present invention provides the use of a diarylheptane compound as shown in Formula I or a pharmaceutically acceptable salt, ester, solvate, or prodrug thereof in the preparation of an NF-κB pathway inhibitor.

[0013] The fourth aspect of the present invention provides the use of a diarylheptane compound as shown in Formula I or a pharmaceutically acceptable salt, ester, solvate, or prodrug thereof in the preparation of a MAPK signaling pathway inhibitor.

[0014] The fifth aspect of the present invention provides the use of a diarylheptane compound as shown in Formula I or a pharmaceutically acceptable salt, ester, solvate, or prodrug thereof in the preparation of an activator of the PI3K / Akt / mTOR signaling pathway.

[0015] In a sixth aspect, the present invention provides the use of a diarylheptane compound as shown in Formula I or a pharmaceutically acceptable salt, ester, solvate, or prodrug thereof in the preparation of an activator of the Nrf2 / HO-1 signaling pathway.

[0016] In some embodiments of the present invention, the pharmaceutically acceptable salt of the diarylheptane compound includes any one of hydrochloride, nitrate, acetate, methanesulfonate, phosphate, citrate, fumarate, sulfate, succinate, tartrate, citrate, hydrobromide, hydroiodide, lactate, tosylate, cinnamate, salicylate, malonate, glutarate, and malate.

[0017] In a seventh aspect, the present invention provides a pharmaceutical composition, the active substance of which includes a diarylheptane compound as shown in Formula I or a pharmaceutically acceptable salt, ester, solvate, or prodrug thereof.

[0018] In some embodiments of the present invention, the active substance of the pharmaceutical composition further comprises an antioxidant. Preferably, the antioxidant is N-Acetylcysteine.

[0019] In some embodiments of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0020] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of a solvent, an excipient, a diluent, a binder, a disintegrant, a dispersant, a flavoring agent, a suspending agent, a surfactant, an isotonic agent, a thickener, an emulsifier, a preservative, a glidant, and a lubricant.

[0021] In some embodiments of the present invention, the dosage form of the drug is any one of tablets, capsules, granules, injections, powder injections, eye drops, liniments, suppositories, ointments, and aerosols.

[0022] In some embodiments of the invention, the excipient comprises water.

[0023] In some embodiments of the present invention, the filler comprises at least one of starch and sucrose.

[0024] In some embodiments of the present invention, the binder includes at least one of a cellulose derivative, alginate, gelatin, and polyvinyl pyrrolidone.

[0025] In some embodiments of the invention, the humectant comprises glycerin.

[0026] In some embodiments of the present invention, the disintegrant comprises at least one of agar, calcium carbonate, and sodium bicarbonate.

[0027] In some embodiments of the invention, the absorption enhancer comprises a quaternary ammonium compound.

[0028] In some embodiments of the invention, the surfactant comprises cetyl alcohol.

[0029] In some embodiments of the present invention, the adsorption carrier includes at least one of kaolin and bentonite.

[0030] In some embodiments of the present invention, the lubricant includes at least one of talc, calcium stearate, magnesium stearate, and polyethylene glycol.

[0031] In some embodiments of the present invention, the dosage form of the pharmaceutical composition is any conventional dosage form in the art.

[0032] In some embodiments of the present invention, the dosage form of the pharmaceutical composition is in the form of solid, semisolid or liquid, and can be an aqueous solution, non-aqueous solution or suspension.

[0033] In some embodiments of the present invention, the dosage form of the pharmaceutical composition is tablets, capsules, soft capsules, granules, pills, oral liquids, dry suspensions, pellets, dry extracts, injections, and infusions.

[0034] It is understood that the pharmaceutical composition of the present invention can be introduced into the body (such as muscle, intradermal, subcutaneous, intravenous, mucosal tissue) by oral administration, injection, spraying, penetration, absorption, physical or chemical mediation; or can be introduced into the body after being mixed or encapsulated with other substances.

[0035] In some embodiments of the present invention, the uses of the pharmaceutical composition include anti-inflammation, anti-oxidation, inhibition of mitochondrial membrane potential, inhibition of reactive oxygen species, inhibition of NF-κB pathway, inhibition of MAPK signaling pathway, activation of PI3K / Akt / mTOR signaling pathway and / or activation of Nrf2 / HO-1 signaling pathway.

[0036] Other features and advantages of the present invention will be set forth in the description which follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0038] Figure 1 The effect of compound DH on the activity of RAW264.7 macrophages; data are expressed as mean±SD (n=3), * : p<0.05vs.LPS group; ** :p<0.01vs.LPS group.

[0039] Figure 2 The effect of compound DH on the release of inflammatory factor NO in RAW264.7 macrophages; data are expressed as mean±SD (n=3), ## : p<0.01vs.control group; * : p<0.05vs.LPS group; ** :p<0.01vs.LPS group.

[0040] Figure 3 The effect of compound DH on the release of inflammatory factors IL-6 and IL-1β; ## : p<0.01vs.control group; ** :p<0.01vs.LPS group.

[0041] Figure 4 is the effect of compound DH on the activity of BMDM macrophages; * :p<0.05vs.LPS group.

[0042] Figure 5 The effect of compound DH on the release of inflammatory factor NO in BMDM macrophages; ## : p<0.01vs.control group; * : p<0.05vs.LPS group; ** :p<0.01vs.LPS group.

[0043] Figure 6 The effect of compound DH on the mRNA and protein expression of iNOS and COX-2 in RAW264.7 macrophages; A is the expression level of iNOS mRNA, B is the expression level of COX-2 mRNA, C is the result of Western blotting, D is the relative expression level of iNOS protein, and E is the relative expression level of COX-2 protein; ## : p<0.01vs.control group; * : p<0.05vs.LPS group; ** :p<0.01vs.LPS group.

[0044] Figure 7The effect of compound DH on the mRNA and protein expression of iNOS in BMDM macrophages; A is the expression level of iNOS mRNA, B is the relative expression level of iNOS protein, and C is the Western blotting result; ## : p<0.01vs.control group; ** :p<0.01vs.LPS group.

[0045] Figure 8 The effect of compound DH on the mRNA expression of MCP-1, CCL2, IL-1β and IL-6 in RAW264.7 macrophages; A is MCP-1, B is CCL2, C is IL-1β, and D is IL-6; ## : p<0.01vs.control group; * : p<0.05vs.LPS group; ** :p<0.01vs.LPS group.

[0046] Fig. 9 The effect of compound DH on the mRNA expression of MCP-1, CCL2, IL-1β and IFN-γ in BMDM macrophages; A is MCP-1, B is CCL2, C is IL-1β, and D is IFN-γ; ## : p<0.01vs.control group; * : p<0.05vs.LPS group; ** :p<0.01vs.LPS group.

[0047] Fig.10 The effect of compound DH on mitochondrial membrane potential in RAW264.7 macrophages was detected using a microplate reader; ## : p<0.01vs.control group; * :p<0.05vs.LPS group.

[0048] Fig.11 The effect of compound DH on mitochondrial membrane potential in RAW264.7 macrophages was photographed using an immunofluorescence microscope;

[0049] Fig.12 The effect of compound DH on reactive oxygen species (ROS) in RAW264.7 macrophages was detected using a microplate reader; # : p<0.05vs.control group; * : p<0.05vs.LPS group; ** :p<0.01vs.LPS group.

[0050] Fig.13The effect of compound DH on reactive oxygen species (ROS) in RAW264.7 macrophages was photographed using an immunofluorescence microscope;

[0051] Fig.14 The effects of the compound DH combined with the antioxidant NAC on the activity of RAW264.7 macrophages, their NO release and inflammatory factors; A is the effect on the NO release of the inflammatory factor, B is the effect on the activity of RAW264.7 macrophages, C is the visualization analysis of the synergistic effect of DH alone and the combined effect of DH and NAC on NO release, and D to G are the effects on the mRNA expression of inflammatory factors iNOS, CCL2, IL-6 and IL-1β, respectively; ## : p<0.01vs.control group; * : p<0.05vs.LPS group; ** : p<0.01vs.LPS group; & : p<0.05vs.DH+NAC group; && :p<0.01vs.DH+NAC group.

[0052] Fig.15 The effect of compound DH on NF-κB pathway in RAW264.7 macrophages; A is the ratio of phosphorylated p65 protein to p65 protein expression, B is the expression of p65 protein in the cytoplasm and nucleus, and C is a fluorescence microscope photo; # : p<0.05vs.control group; ## : p<0.01vs.control group; * : p<0.05vs.LPS group; ** :p<0.01vs.LPS group.

[0053] Fig.16 The effect of compound DH on the MAPK pathway in RAW264.7 macrophages; A is the Western blotting result, B is the p38 protein in the MAPK pathway, C is the JNK protein in the MAPK pathway, and D is the ERK protein in the MAPK pathway; ## : p<0.01vs.control group; * : p<0.05vs.LPS group; ** :p<0.01vs.LPS group.

[0054] Fig.17The effect of the combination of compound DH and JNK agonist Anisomycin on NO release in RAW264.7 macrophages; A is the effect of the combination of DH and Anisomycin on the release of inflammatory factor NO, and B is the effect of the combination of DH and Anisomycin on the activity of RAW264.7 macrophages; ## : p<0.01vs.control group; ** :p<0.01vs.LPS group.

[0055] Fig.18 The effect of compound DH on the PI3K pathway in RAW264.7 macrophages; A is the AKT protein in the PI3K pathway, B is the mTOR protein in the PI3K pathway, C is the p70S6K protein in the PI3K pathway, and D is the 4EBP1 in the PI3K pathway; E is the Western blotting result; ## : p<0.01vs.control group; * : p<0.05vs.LPS group; ** :p<0.01vs.LPS group.

[0056] Fig.19 The effects of the combination of compound DH and Wortmanin, an inhibitor of the PI3K pathway, on NO and iNOS, COX-2, IL-6, IL-1β and MCP-1 in RAW264.7 macrophages; A is the effect of the combination of DH and Wortmanin on the release of inflammatory factor NO, B is the effect of the combination of DH and Wortmanin on the activity of RAW264.7 macrophages, and C to G are the effects of the combination of DH and Wortmanin on the mRNA expression of iNOS, COX-2, IL-6, IL-1β and MCP-1, respectively; ## : p<0.01vs.control group; * : p<0.05vs.LPS group; ** : p<0.01vs.LPS group; & :p<0.05vs.DH+wortmanin group.

[0057] Fig. 20The effects of the combination of compound DH and LY294002, an inhibitor of the PI3K pathway, on NO and iNOS, COX-2, IL-6, IL-1β and MCP-1 in RAW264.7 macrophages; A is the effect of the combination of DH and LY294002 on the release of inflammatory factor NO, B is the effect of the combination of DH and LY294002 on the activity of RAW264.7 macrophages, and C to G are the effects of the combination of DH and LY294002 on the mRNA expression of iNOS, COX-2, IL-6, IL-1β and MCP-1, respectively; ## : p<0.01vs.control group; * : p<0.05vs.LPS group; ** : p<0.01vs.LPS group; & : p<0.05vs.DH+LY294002 group; && :p<0.01vs.DH+LY294002 group.

[0058] Fig.21 The effect of compound DH on the Nrf2 / HO-1 pathway in RAW264.7 macrophages; A is the Western blotting result, B is the Nrf2 protein in the Nrf2 / HO-1 pathway, C is the HO-1 protein in the Nrf2 / HO-1 pathway, and D is the fluorescence microscope photo of Nrf2; ## : p<0.01vs.control group; * : p<0.05vs.LPS group; ** :p<0.01vs.LPS group.

[0059] Fig. 22 The effect of the combination of compound DH and antioxidant NAC on the Nrf2 / HO-1 pathway in RAW264.7 macrophages; A is the Western blotting result, B is the Nrf2 protein, and C is the HO-1 protein; ## : p<0.01vs.control group; * : p<0.05vs.LPS group; ** : p<0.01vs.LPS group; & : p<0.05vs.DH+NAC group; && :p<0.01vs.DH+NAC group.

[0060] Fig.23The effect of the combination of compound DH and antioxidant NAC on the Nrf2 / HO-1 pathway in BMDM macrophages; A is the Western blotting result, B is Nrf2 in the Nrf2 / HO-1 pathway, C is the HO-1 protein in the Nrf2 / HO-1 pathway, and D is a fluorescence microscope photo of Nrf2; ## : p<0.01vs.control group; * : p<0.05vs.LPS group; ** : p<0.01vs.LPS group; & :p<0.05vs.DH+NAC group.

[0061] Fig.24 The figures show the effects of the combination of compound DH and Nrf2 agonist THBQ on NO and iNOS, IL-6, MCP-1 and CCL2 in RAW264.7 macrophages; A shows the effect of the combination of DH and THBQ on the release of inflammatory factor NO, B shows the effect of the combination of DH and THBQ on the activity of RAW264.7 macrophages, and C to F show the effects of the combination of DH and THBQ on the mRNA expression of iNOS, IL-6, MCP-1 and CCL2. # : p<0.05vs.control group; ## : p<0.01vs.control group; * : p<0.05vs.LPS group; ** : p<0.01vs.LPS group; & : p<0.05vs.DH+TBHQ group; && :p<0.01vs.DH+TBHQ group. DETAILED DESCRIPTION

[0062] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0063] The words "preferably", "more preferably", etc. in the present invention refer to embodiments of the present invention that may provide certain beneficial effects in certain circumstances. However, other embodiments may also be preferred under the same circumstances or other circumstances. In addition, the description of one or more preferred embodiments does not imply that other embodiments are not applicable, nor is it intended to exclude other embodiments from the scope of the present invention.

[0064] When a numerical range is disclosed herein, the above range is considered to be continuous and includes the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0065] In the description of the present invention, the reference term "and / or" includes all and any combinations of one or more of the associated listed items.

[0066] In the description of the present invention, the description with reference to the terms "one embodiment", "some embodiments", "illustrative embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0067] If not otherwise specified, "DMEM complete medium" herein refers to DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin mixture. "1640 complete medium" herein refers to 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin mixture.

[0068] RAW264.7 cells were cultured and passaged using DMEM complete medium. BMDM cells were taken from the bone marrow between the femur and tibia of BALB / c mice and cultured in 1640 complete medium containing 12.5ng / mL M-CSF. On the 3rd day, fresh 1640 complete medium containing 6.25ng / mL M-CSF was replaced. On the 5th day, fresh 1640 complete medium was replaced. After 7 days of adherent culture, bone marrow-derived precursor cells differentiated into BMDM macrophages.

[0069] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0070] Example 1: Synthesis of Compound DH

[0071] This example provides a diarylheptane compound (hereinafter referred to as compound DH), whose chemical structure is shown in Formula I;

[0072]

[0073] The compound DH is extracted from plants, and the specific methods of obtaining it include:

[0074] The dried plant rhizomes were extracted with 75% ethanol for 3 times, each time for 2.5 h, and concentrated under reduced pressure until there was no alcohol taste to obtain 30 L of water-suspended extract, which was then extracted 4 times with equal volumes of cyclohexane, chloroform, ethyl acetate and n-butanol to obtain cyclohexane layer, chloroform layer, ethyl acetate layer and n-butanol layer, respectively, and then separated by chromatography to obtain compound DH.

[0075] Experimental Example 2: Effects on the activity of RAW264.7 macrophages

[0076] This example detected the effects of different concentrations of the above compound DH on the activity of RAW264.7 macrophages, and the specific method was as follows:

[0077] RAW264.7 macrophages in the logarithmic growth phase were inoculated into a 96-well plate, and 100 μL of cell suspension was added to each well. The cell density was 2.5×10 4 / mL, with 3 replicates per group, and cultured in a constant temperature incubator at 37°C and 5% CO2. After cell attachment, 300ng / mL of LPS was added to 6 groups of mouse RAW264.7 macrophages, and DH solutions with DMSO as solvent and concentrations of 0μM, 6.25μM, 12.5μM, 25μM, 50μM and 100μM were added for co-incubation as the test drug groups, where 0μM means that DH was not added, and only 300ng / mL of LPS was added; in addition, RAW264.7 macrophages without LPS and DH were used as blank control groups.

[0078] After 24 hours of co-incubation, 10 μL of MTT reagent was added to each well and cultured at 37°C for 4 hours. DMSO was added and shaken on a shaker for 10 minutes. The absorbance of each well was measured at 490 nm using an enzyme reader to detect cell activity.

[0079] Test results such as Figure 1 As shown, it was shown that compound DH was non-toxic to RAW264.7 macrophages in the range of 6.25-100 μM, and had a certain therapeutic effect on the reduction of LPS-induced RAW264.7 macrophage activity.

[0080] Experimental Example 3: Anti-inflammatory activity detection based on RAW264.7 macrophages

[0081] This example detected the effects of different concentrations of the above compound DH on the anti-inflammatory activity of RAW264.7 macrophages, and the specific method was as follows:

[0082] RAW264.7 macrophages in the logarithmic growth phase were inoculated into a 96-well plate, and 100 μL of cell suspension was added to each well. The cell density was 2.5×10 4 / mL, with 3 replicates per group, and cultured in a constant temperature incubator at 37°C and 5% CO2. After cell attachment, 300ng / mL of LPS was added to 6 groups of mouse RAW264.7 macrophages, and DMSO was used as the solvent, and DH solutions with concentrations of 0μM, 6.25μM, 12.5μM, 25μM, 50μM and 100μM were added for co-incubation as the test drug group, where 0μM means that DH was not added, and only 300ng / mL of LPS was added; in addition, RAW264.7 macrophages without LPS and DH were used as blank control groups. After 24 hours of co-incubation, the release of NO, IL-6 and IL-1β in cells was detected.

[0083] Test results such as Figure 2 and Figure 3 As shown, Figure 2 The results show the effect of compound DH on the release of inflammatory factor NO in RAW264.7 macrophages. Figure 3 The above results show that compound DH can significantly inhibit the release of NO, IL-6 and IL-1β in RAW264.7 macrophages, and it is dose-dependent, which indicates that compound DH of the present invention has a good inhibitory effect on inflammatory factors NO, IL-6 and IL-1β, and can be used to prepare anti-inflammatory drugs.

[0084] Experimental Example 4: Effect on BMDM macrophage activity

[0085] This example detects the effects of different concentrations of the above compound DH on the activity of mouse BMDM macrophages, and the specific method is as follows:

[0086] The femur and tibia of BALB / c mice were removed and the ends of the bones were trimmed. The tissue was washed with 1640 complete medium. The obtained tissue blocks were filtered through a 70μm cell sieve and the cells were recovered by centrifugation. The cells were then cultured in 1640 complete medium containing 12.5ng / mL M-CSF. On the 3rd day, fresh 1640 complete medium containing 6.25ng / mL M-CSF was replaced. On the 5th day, fresh 1640 complete medium was replaced. After 7 days of adherent culture, the bone marrow-derived precursor cells differentiated into BMDM macrophages. After cell attachment, 800ng / mL LPS was added to 6 groups of mouse BMDM macrophages, and DMSO was used as solvent, and DH solution with concentrations of 0μM, 6.25μM, 12.5μM, 25μM, 50μM and 100μM was added for co-incubation as the test drug group, where 0μM means no DH was added, only 800ng / mL LPS was added; in addition, mouse BMDM macrophages without LPS and DH were used as blank control group. After 24 hours of co-incubation, the cell survival rate was detected by MTT method.

[0087] Test results such as Figure 4 As shown, it was shown that compound DH had no effect on the activity of BMDM macrophages in the range of 6.25-100 μM, and had a certain therapeutic effect on the reduction of LPS-induced BMDM macrophage activity.

[0088] Experimental Example 5: Anti-inflammatory activity detection based on BMDM macrophages

[0089] This example detects the effects of different concentrations of the above compound DH on the anti-inflammatory activity of BMDM macrophages, and the specific method is as follows:

[0090] The femur and tibia of BALB / c mice were removed and the ends of the bones were trimmed. The tissue was washed with 1640 complete medium. The obtained tissue blocks were filtered through a 70μm cell sieve and the cells were recovered by centrifugation. The cells were then cultured in 1640 complete medium containing 12.5ng / mL M-CSF. On the 3rd day, fresh 1640 complete medium containing 6.25ng / mL M-CSF was replaced. On the 5th day, fresh 1640 complete medium was replaced. After 7 days of adherent culture, the bone marrow-derived precursor cells differentiated into BMDM macrophages. After cell attachment, 800ng / mL LPS was added to 6 groups of mouse BMDM macrophages, and DMSO was used as solvent, and DH solution with concentrations of 0μM, 6.25μM, 12.5μM, 25μM, 50μM and 100μM was added for co-incubation as the test drug group, where 0μM means no DH was added, only 800ng / mL LPS was added; in addition, mouse BMDM macrophages without LPS and DH were used as blank control group. After 24 hours of co-incubation, the release of cellular NO was detected.

[0091] Test results such as Figure 5 As shown, it is shown that compound DH can significantly inhibit the release of NO in BMDM macrophages and presents a dose-dependency, which indicates that compound DH of the present invention has a good inhibitory effect on the inflammatory factor NO in BMDM cells and can be used to prepare anti-inflammatory drugs.

[0092] Experimental Example 6: Effects on the expression of iNOS and COX-2 in RAW264.7 macrophages

[0093] This example detected the effects of different concentrations of the above compound DH on the expression of iNOS and COX-2 in RAW264.7 macrophages. The specific method is as follows:

[0094] Mouse RAW264.7 macrophages were inoculated into 6-well plates, and 2 mL of cell suspension was added to each well. The cell density was 1×10 6 / mL, and cultured overnight in a constant temperature incubator at 37°C and 5% CO2. After cell attachment, 300ng / mL of LPS was added to 5 groups of mouse RAW264.7 macrophages, and DMSO was used as the solvent, and 12.5μM, 25μM, and 50μM DH solutions were added for co-incubation as the test drug group; in addition, macrophages added with LPS and 40μM commercial anti-inflammatory drug indometacin were used as the control group, mouse RAW264.7 macrophages without LPS and compound DH were used as the blank control group, and mouse RAW264.7 macrophages added with only LPS were used as the model group. After 24 hours of co-incubation, the mRNA and protein expression of iNOS and COX-2 were detected by qRT-PCR and Western blotting methods, respectively.

[0095] GAPDH was used as an internal reference during the detection process. The primary antibodies GAPDH and COX-2 were purchased from Wuhan Tri-Tac (Wuhan, China), and iNOS was purchased from Abcam (Shanghai, China). The secondary antibodies were rabbit and mouse antibodies, both purchased from Cell Signaling Technology (Danvers, USA). The primary antibodies GAPDH and iNOS were diluted at a ratio of 1:5000, COX-2 was diluted at a ratio of 1:2000, and the secondary antibody was diluted at a ratio of 1:2000 before use.

[0096] Test results such as Figure 6 As shown, A and B are the mRNA expression levels of iNOS and COX-2, respectively, C is the protein expression level of iNOS and COX-2, D and E are the protein statistical results of iNOS and COX-2. The results show that compound DH can down-regulate the expression of iNOS and COX-2 in RAW264.7 macrophages at the mRNA transcription and protein levels, and the inhibitory effect of DH on the protein expression of iNOS and COX-2 in the concentration range of 25-50 μM can reach or even exceed the effect level of 40 μMindometacin, indicating that compound DH has a very good inhibitory effect on LPS-induced inflammation.

[0097] Experimental Example 7: Effect on iNOS expression in BMDM macrophages

[0098] This example detects the effects of different concentrations of the above compound DH on the expression of iNOS in BMDM macrophages, and the specific method is as follows:

[0099] The femur and tibia of BALB / c mice were removed and the ends of the bones were trimmed. The tissue was washed with 1640 complete medium. The obtained tissue blocks were filtered through a 70μm cell sieve and the cells were recovered by centrifugation. The cells were then cultured in 1640 complete medium containing 12.5ng / mL M-CSF. On the 3rd day, fresh 1640 complete medium containing 6.25ng / mL M-CSF was replaced. On the 5th day, fresh 1640 complete medium was replaced. After 7 days of adherent culture, the bone marrow-derived precursor cells differentiated into BMDM macrophages. After cell attachment, 800ng / mL LPS was added to 5 groups of mouse BMDM macrophages, and DMSO was used as solvent, and DH solution with concentrations of 12.5μM, 25μM, and 50μM was added for co-incubation as the test drug group; in addition, macrophages added with LPS and 40μM commercial anti-inflammatory drug indometacin were used as the control group, mouse BMDM macrophages without LPS and DH were used as the blank control group, and mouse BMDM macrophages added with only LPS were used as the model group. After 24 hours of co-incubation, the mRNA and protein expression of iNOS were detected by qRT-PCR and Western blotting methods, respectively.

[0100] βaction was used as an internal reference during the detection process. The primary antibody βaction was purchased from Wuhan Tri-Tek Co., Ltd. (Wuhan, China), and iNOS was purchased from Abcam (Shanghai, China). The secondary antibodies were rabbit and mouse antibodies, both purchased from Cell Signaling Technology (Danvers, USA). The primary antibodies βaction and iNOS were diluted at a ratio of 1:5000, and the secondary antibody was diluted at a ratio of 1:2000 before use.

[0101] Test results such as Figure 7 As shown by Figure 7 It can be seen that compound DH can downregulate the expression of iNOS in BMDM macrophages at the mRNA transcription and protein levels, and the inhibitory effect of DH on the protein expression of iNOS in the concentration range of 25-50 μM is far superior to the effect level of 40 μM indometacin, indicating that DH has a very good inhibitory effect on inflammation.

[0102] Experimental Example 8: Effects on inflammatory factors in RAW264.7 macrophages

[0103] This example detects the effects of different concentrations of the above compound DH on the expression of inflammatory factors in RAW264.7 macrophages. The specific method is as follows:

[0104] Mouse RAW264.7 macrophages were inoculated into 12-well plates, and 1 mL of cell suspension was added to each well. The cell density was 3×105 / mL, and cultured overnight in a constant temperature incubator at 37℃ and 5% CO2. After cell attachment, 300ng / mL of LPS was added to 5 groups of mouse RAW264.7 macrophages, and DMSO was used as the solvent, and DH solutions with concentrations of 12.5μM, 25μM, and 50μM were added for co-incubation as the test drug group; in addition, RAW264.7 cells with LPS and 40μM commercial anti-inflammatory drug indometacin were added as the control group, mouse RAW264.7 macrophages without LPS and DH were used as the blank control group, and mouse RAW264.7 macrophages with only LPS were used as the model group. After 12 hours of co-incubation, the mRNA expression of inflammatory factors MCP-1, CCL2, IL-1β, and IL-6 was detected by qRT-PCR.

[0105] Test results such as Figure 8 As shown, it is shown that compound DH can significantly down-regulate the expression of mRNA of inflammatory factors MCP-1, CCL2, IL-1β and IL-6 in RAW264.7 macrophages, and the inhibitory effect of compound DH on the mRNA of MCP-1, CCL2 and IL-1β at a concentration of about 50 μM is much better than the inhibitory level of 40 μM indometacin, indicating that the compound DH of the present invention has a significant inhibitory effect on LPS-induced RAW264.7 macrophage inflammation and can be used to prepare anti-inflammatory drugs.

[0106] Experimental Example 9: Effects on inflammatory factors in BMDM macrophages

[0107] This example detects the effects of different concentrations of the above compound DH on the expression of inflammatory factors in BMDM macrophages, and the specific method is as follows:

[0108] The femur and tibia of BALB / c mice were removed and the ends of the bones were trimmed. The tissue was washed with 1640 complete medium. The obtained tissue blocks were filtered through a 70μm cell sieve and the cells were recovered by centrifugation. The cells were then cultured in 1640 complete medium containing 12.5ng / mL M-CSF. On the 3rd day, fresh 1640 complete medium containing 6.25ng / mL M-CSF was replaced. On the 5th day, fresh 1640 complete medium was replaced. After 7 days of adherent culture, the bone marrow-derived precursor cells differentiated into BMDM macrophages. 800ng / mL LPS was added to 5 groups of mouse BMDM macrophages, and DMSO was used as solvent, and DH solution with concentrations of 12.5μM, 25μM, and 50μM was added for co-incubation as the test drug group; in addition, macrophages added with LPS and 40μM commercial anti-inflammatory drug indometacin were used as the control group, mouse BMDM macrophages without LPS and DH were used as the blank control group, and mouse BMDM macrophages added with only LPS were used as the model group. After 12 hours of co-incubation, the mRNA expression of inflammatory factors IFN-γ, MCP-1, CCL2, and L-1β was detected by qRT-PCR.

[0109] Test results such as Fig. 9 As shown, A is the detection result of mRNA expression of inflammatory factor IFN-γ, B is the detection result of mRNA expression of MCP-1, C and D are the detection results of mRNA expression of CCL2 and L-1β, respectively. The results show that compound DH can significantly down-regulate the expression of mRNA of inflammatory factors IFN-γ, MCP-1, CCL2 and IL-1β in BMDM macrophages, indicating that the compound DH of the present invention has a significant inhibitory effect on LPS-induced BMDM macrophage inflammation and can be used to prepare anti-inflammatory drugs.

[0110] Experimental Example 10: Effects on LPS-induced mitochondrial membrane potential

[0111] This example detected the effects of different concentrations of the above compound DH on the mitochondrial membrane potential of RAW264.7 macrophages induced by LPS, and the specific method was as follows:

[0112] RAW264.7 cells (2.5×10 4The cells were cultured overnight in a 96-well plate (100 μL culture medium / well), and then treated with 12.5 μM, 25 μM, and 50 μM DH for 4 hours in the presence of 300 ng / mL LPS. The cells were fixed with 4% paraformaldehyde for 15 minutes. The cells were then treated with JC-1 working solution at 37°C for 20 minutes and washed twice with JC-1 buffer. Images were taken using a fluorescence microscope. Fluorescence intensity was measured using a fluorescence microplate reader.

[0113] Note: At higher mitochondrial membrane potential, JC-1 aggregates in the mitochondrial matrix to form polymers, emitting red fluorescence. When the mitochondrial membrane potential decreases, JC-1 remains in the mitochondrial matrix as a monomer, emitting green fluorescence.

[0114] Test results such as Fig.10 and Fig.11 As shown, Fig.10 The effect of compound DH on mitochondrial membrane potential in RAW264.7 macrophages. Fig.11 The corresponding fluorescence staining observation diagram is shown in Figure 2. The results showed that LPS stimulation caused a significant decrease in the mitochondrial membrane potential (MMP) level. After 4 hours of DH treatment, the MMP level gradually increased (e.g. Fig.10 shown).

[0115] In addition, the RAW264.7 cells in the LPS group emitted green fluorescence, indicating that the mitochondrial membrane potential was significantly reduced by LPS. As the DH treatment time increased, the color of the RAW264.7 cells changed from green to red, indicating that DH gradually restored the mitochondrial membrane potential reduced by LPS. CCCP was used as a positive control to induce mitochondrial membrane potential depolarization. The CCCP group showed green fluorescence in the immunofluorescence images, confirming the effectiveness of the experimental system (e.g. Fig.11 shown).

[0116] The above results indicate that the compound DH of the present invention has a significant reversal effect on the decrease of mitochondrial membrane potential induced by LPS, and can be used to prepare antioxidant drugs.

[0117] Experimental Example 11: Effects on ROS in RAW264.7 cells

[0118] This example detected the effects of different concentrations of the above compound DH on ROS induced by LPS in RAW264.7 macrophages. The specific method is as follows:

[0119] RAW264.7 cells (2.5×10 4 / well, 100μL culture medium / well) were cultured overnight in a 96-well plate, and then 12.5μM, 25μM, 50μM DH, 40μM indomethacin, 50μM H2O2, 50μM H2O2 and 25μM DH were applied to RAW264.7 cells for 4 hours in the presence of 300ng / mL LPS. Mouse RAW264.7 macrophages without LPS and DH were used as blank controls. After that, 4% paraformaldehyde was added for fixation for 15 minutes. Next, the cells were treated with JC-1 working solution at 37°C for 20 minutes and washed twice with JC-1 buffer. Images were taken using a fluorescence microscope. Fluorescence intensity was measured using a fluorescence microplate reader. Note: DCFH is oxidized by ROS in the cell and converted into DCF, emitting strong green fluorescence.

[0120] Test results such as Fig.12 and Fig.13 As shown. Fig.12 The results shown indicate that LPS stimulation resulted in a significant increase in ROS levels, which were significantly reduced after 4 hours of treatment with different concentrations of compound DH. Fig.13 The changes in ROS in RAW264.7 cells can be observed more directly under a microscope. LPS significantly increased the production of ROS in RAW264.7 cells, emitting strong green fluorescence. After treatment with different concentrations of DH, the green fluorescence decreased, indicating that DH reversed the effect of LPS on RAW264.7 cells. When 50 μM H2O2 was added, the green fluorescence in RAW264.7 cells increased. This phenomenon indicates that ROS production in RAW264.7 cells increased. However, when 50 μM H2O2 and 50 μM DH were co-administered, the green fluorescence disappeared, further confirming that DH inhibited ROS production in RAW264.7 cells.

[0121] The above phenomena indicate that compound DH has a good inhibitory effect on ROS and can be used to prepare antioxidant drugs.

[0122] Experimental Example 12: Detection of the effect of compound DH combined with antioxidants

[0123] This example detects the effects of the above compound DH combined with the antioxidant N-Acetylcysteine ​​(NAC) on the NO release, survival rate and inflammatory factor expression of RAW264.7 macrophages induced by LPS. The specific method is as follows:

[0124] 1. Effects on NO release from RAW264.7 macrophages induced by LPS:

[0125] RAW264.7 macrophages in the logarithmic growth phase were inoculated into a 96-well plate, and 100 μL of cell suspension was added to each well. The cell density was 2.5×104 / mL, with 3 replicates per group, and cultured in a constant temperature incubator at 37°C and 5% CO2. After cell attachment, 300ng / mL of LPS was added to 6 groups of mouse RAW264.7 macrophages, and DMSO was used as the solvent, with DH solutions at concentrations of 0μM, 12.5μM, 25μM and 50μM, and / or 100μM NAC were added for co-incubation as the test drug group, where 0μM means that no compound DH was added, only 300ng / mL of LPS and antioxidant NAC were added; in addition, RAW264.7 macrophages without LPS, DH or NAC were used as blank control groups. In addition, during the drug administration process, NAC was administered 4 hours in advance. After 24 hours of co-incubation, the release of cellular NO was detected.

[0126] 2. Effect on the survival rate of LPS-induced RAW264.7 macrophages:

[0127] RAW264.7 macrophages in the logarithmic growth phase were inoculated into a 96-well plate, and 100 μL of cell suspension was added to each well. The cell density was 2.5×10 4 / mL, with 3 replicates per group, and cultured in a constant temperature incubator at 37°C and 5% CO2. After cell attachment, 300ng / mL of LPS was added to 6 groups of mouse RAW264.7 macrophages, and DMSO was used as the solvent, with concentrations of 0μM, 12.5μM, 25μM and 50μM DH solution, and / or 100μM NAC were added for co-incubation as the test drug group, where 0μM means that no compound DH was added, only 300ng / mL of LPS and antioxidant NAC were added; in addition, RAW264.7 macrophages without LPS, DH or NAC were used as blank control groups. In addition, during the drug administration process, NAC was administered 4 hours in advance. After 24 hours of co-incubation, 10μL of MTT reagent was added to each well, cultured at 37°C for 4 hours, DMSO was added and shaken on a shaker for 10 minutes, and the absorbance of each well was measured at 490nm using an enzyme reader to detect cell activity.

[0128] 3. Effects on the expression of inflammatory factors in RAW264.7 macrophages induced by LPS:

[0129] RAW264.7 macrophages were inoculated into 12-well plates, and 1 mL of cell suspension was added to each well. The cell density was 3×10 5 / mL, and cultured overnight in a constant temperature incubator at 37℃ and 5% CO2. After cell attachment, 300ng / mL LPS was added to 6 groups of mouse RAW264.7 macrophages, and DMSO was used as solvent, DH solution with a concentration of 25, and or 100μM NAC were added for co-incubation as the test drug group; in addition, RAW264.7 cells added with LPS and 40μM commercial anti-inflammatory drug indometacin were used as the control group, RAW264.7 macrophages without LPS, compound DH and NAC were used as the blank control group, and mouse RAW264.7 macrophages added with only LPS were used as the model group. After 12 hours of co-incubation, the mRNA expression of inflammatory factors iNOS, CCL2, IL-6 and IL-1β was detected by qRT-PCR.

[0130] The above test results are as follows Fig.14 As shown, A is the test result of the effect on the NO release of RAW264.7 macrophages induced by LPS, B is the test result of the effect on the survival rate of RAW264.7 macrophages induced by LPS, C is the visualization analysis of the synergistic effect of DH alone and DH and NAC on NO release, and D~G are the results of inflammatory factor expression detection. The results showed that the combined treatment of 50μM DH and 100μM NAC in RAW264.7 cells better inhibited the release of NO than the treatment with 50μM DH alone, and had no obvious cytotoxicity to RAW264.7 cells. The visualization analysis result of the synergistic effect of the combined action of DH and NAC on NO release was 35.42, which was much greater than 10, indicating that the anti-inflammatory effect of the combined action of DH and NAC was synergistic. Further exploration also found that the combined treatment of 50μM DH and 100μM NAC better reduced the expression of inflammatory cytokines iNOS, CCL2, IL-6 and IL-1βmRNA than the treatment with 50μM DH alone.

[0131] The above results indicate that compound DH can relieve inflammation by inhibiting oxidative stress, and the effect is better when used in combination with the antioxidant NAC, and it can be used to prepare antioxidant and anti-inflammatory drugs.

[0132] Experimental Example 13: Effects on LPS-induced NF-κB pathway in RAW264.7 macrophages

[0133] This example detected the effects of different concentrations of the above compound DH on the NF-κB pathway of RAW264.7 macrophages induced by LPS, and the specific method was as follows:

[0134] Mouse RAW264.7 macrophages were inoculated into 6-well plates, and 2000 μL of cell suspension was added to each well. The cell density was 3×104 / mL, and cultured overnight in a constant temperature incubator at 37°C and 5% CO2. After the cells adhered, 12.5μM, 25μM and 50μM DH solutions and 300μg / mL LPS were added and incubated for 4 hours as the test drug group, and mouse RAW264.7 macrophages without LPS and DH were used as the blank control group, and mouse RAW264.7 macrophages with only LPS added were used as the model group. The effect of nuclear factor p65 nuclear translocation was observed by fluorescence microscopy, and the expression of p65 and phosphorylated p65 as well as p65 protein in the cytoplasm and nucleus was detected by Western blotting.

[0135] GAPDH and LaminB1 were used as internal references during the detection process. The primary antibody GAPDH was purchased from Wuhan Tri-Takahashi (Wuhan, China), and NF-κB p65 and p–NF–κB p65S were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The secondary antibodies were rabbit and mouse antibodies, both purchased from Cell Signaling Technology (Danvers, USA). The primary antibody GAPDH was diluted at a ratio of 1:5000, LaminB1 was diluted at a ratio of 1:2000, NF-κB p65 and p–NF–κB p65S were diluted at a ratio of 1:1000, and the secondary antibody was diluted at a ratio of 1:2000 before use.

[0136] Test results such as Fig.15 As shown, compound DH significantly reduced the ratio of phosphorylated p65 and p65 in RAW264.7 macrophages, inhibited the nuclear translocation of nuclear transcription factor p65, and prevented the activation of NF-κB pathway, indicating that DH has a very good inhibitory effect on oxidative stress and inflammation.

[0137] Experimental Example 14: Effects on LPS-induced MAPK pathway in RAW264.7 macrophages

[0138] This example detects the effect of the above compound DH on the phosphorylation of p38, ERK and JNK proteins in the MAPK pathway of RAW264.7 macrophages. The specific method is as follows:

[0139] Mouse RAW264.7 macrophages were inoculated into 6-well plates, and 2 mL of cell suspension was added to each well. The cell density was 1×10 6100 μg / well, cultured overnight in a constant temperature incubator at 37°C and 5% CO2. After the cells adhered to the wall, 300 ng / mL of LPS was added, and 12.5 μM, 25 μM, and 50 μM DH solutions were added to the cells for co-incubation as the test drug group; in addition, RAW264.7 cells added with LPS and 40 μM commercial anti-inflammatory drug indometacin were used as the control group, mouse RAW264.7 macrophages without LPS and DH were used as the blank control group, and mouse RAW264.7 macrophages only with LPS were used as the model group. After 12 hours of co-incubation, the phosphorylation levels of ERK, p38, and JNK proteins in the MAPK pathway were detected by Western blotting.

[0140] GAPDH was used as an internal reference during the detection process. The primary antibody GAPDH was purchased from Wuhan Tri-Tac (Wuhan, China). The rabbit and mouse antibodies used for JNK, p-JNK, ERK, p-ERK, p38, p-p38 and secondary antibodies were purchased from Cell Signaling Technology (Danvers, USA). The primary antibody GAPDH was diluted at a ratio of 1:5000, the JNK, p-JNK, ERK, p-ERK, p38 and p-p38 were diluted at a ratio of 1:1000, and the secondary antibody was diluted at a ratio of 1:2000 before use.

[0141] Test results such as Fig.16 As shown, compound DH significantly downregulated the phosphorylation of three proteins, p38, ERK and JNK, in the MAPK pathway in RAW264.7 macrophages, and inhibited the activation of the MAPK pathway, indicating that compound DH has a very good inhibitory effect on oxidative stress and inflammation.

[0142] Experimental Example 15: Detection of the combined effect of compound DH and Anisomycin

[0143] This example detected the effect of the above compound DH combined with the JNK agonist Anisomycin on the NO release and survival rate of RAW264.7 macrophages induced by LPS. The specific method is as follows:

[0144] 1. Effects on NO release from RAW264.7 macrophages induced by LPS:

[0145] RAW264.7 macrophages in the logarithmic growth phase were inoculated into a 96-well plate, and 100 μL of cell suspension was added to each well. The cell density was 2.5×10 4 / mL, with 3 replicates per group, and cultured in a constant temperature incubator at 37°C and 5% CO2. After cell attachment, 300ng / mL of LPS was added to 6 groups of mouse RAW264.7 macrophages, and DMSO was used as the solvent, with DH solutions at concentrations of 0μM, 12.5μM, 25μM and 50μM, and / or 10nM Anisomycin were added for co-incubation as the test drug group, where 0μM means that no compound DH was added, only 300ng / mL of LPS and Anisomycin were added; in addition, RAW264.7 macrophages without LPS, DH or Anisomycin were used as blank control groups. In addition, during the drug administration process, Anisomycin was administered 3 hours in advance. After 24 hours of co-incubation, the release of cellular NO was detected.

[0146] 2. Effect on the survival rate of LPS-induced RAW264.7 macrophages:

[0147] RAW264.7 macrophages in the logarithmic growth phase were inoculated into a 96-well plate, and 100 μL of cell suspension was added to each well. The cell density was 2.5×10 4 / mL, with 3 replicates per group, and cultured in a constant temperature incubator at 37°C and 5% CO2. After the cells adhered to the wall, 300ng / mL of LPS was added to 6 groups of mouse RAW264.7 macrophages, and DMSO was used as the solvent, with DH solutions at concentrations of 0μM, 12.5μM, 25μM and 50μM, and / or 10nM Anisomycin were added for co-incubation as the test drug group, where 0μM means that no compound DH was added, only 300ng / mL of LPS and Anisomycin were added; in addition, RAW264.7 macrophages without LPS, DH or Anisomycin were used as blank control groups. In addition, during the administration process, Anisomycin was administered 3 hours in advance. After 24 hours of co-incubation, 10 μL of MTT reagent was added to each well and cultured at 37°C for 4 hours. DMSO was added and shaken on a shaker for 10 minutes. The absorbance of each well was measured at 490 nm using an enzyme reader to detect cell activity.

[0148] Test results such as Fig.17 As shown, A is the NO release test result, B is the cell activity test result, Fig.17 It can be seen that the effect of DH combined with Anisomycin on NO release in RAW264.7 macrophages was not significantly different from that of DH alone. These results indicate that compound DH inhibits the activation of the MAPK signaling pathway, thereby contributing to its antioxidant and anti-inflammatory effects, although this effect is not completely dependent on the MAPK signaling pathway.

[0149] Experimental Example 16: Effects on LPS-induced PI3K pathway in RAW264.7 macrophages

[0150] This example detected the effect of the above compound DH on the phosphorylation of AKT, mTOR, p70S6K and 4EBP1 proteins in the PI3K pathway of RAW264.7 macrophages induced by LPS, and the specific method was as follows:

[0151] Mouse RAW264.7 macrophages were inoculated into 6-well plates, and 2 mL of cell suspension was added to each well. The cell density was 1×10 6 / well, and cultured overnight in a constant temperature incubator at 37°C and 5% CO2. After the cells adhered to the wall, 300ng / mL of LPS was added, and 12.5μM, 25μM, and 50μM DH solutions were added to the groups for co-incubation as the test drug group; in addition, RAW264.7 cells with LPS and 40μM commercial anti-inflammatory drug indometacin were added as the control group, mouse RAW264.7 macrophages without LPS and DH were used as the blank control group, and mouse RAW264.7 macrophages with only LPS were used as the model group. After 24 hours of co-incubation, the phosphorylation levels of AKT, mTOR, p70S6K, and 4EBP1 proteins in the PI3K pathway were detected by Western blotting.

[0152] GAPDH was used as an internal reference during the detection process. The primary antibody GAPDH was purchased from Wuhan Tri-Tek Co., Ltd. (Wuhan, China). The rabbit and mouse antibodies used for mTOR, p-mTOR, AKT, p-AKT, p70S6K, p-p70S6K, p-4EBP1 and the secondary antibodies were purchased from Cell Signaling Technology (Danvers, USA). The primary antibody GAPDH was diluted at a ratio of 1:5000, the mTOR, p-mTOR, AKT, p-AKT, p70S6K, p-p70S6K and p-4EBP1 were diluted at a ratio of 1:1000, and the secondary antibody was diluted at a ratio of 1:2000 before use.

[0153] Test results such as Fig.18 As shown, compound DH has a significant stimulatory effect on the phosphorylation of AKT, mTOR, and p70S6K proteins in the PI3K pathway in RAW264.7 macrophages, and exhibits a significant inhibitory effect on the phosphorylation of 4EBP1 protein. This indicates that DH produces a negative feedback regulation on the PI3K pathway, which can inhibit the activation of MAPK and NF-κB, and play an antioxidant and anti-inflammatory role.

[0154] Experimental Example 17: Detection of the combined effect of compound DH and PI3K pathway inhibitor

[0155] This example detected the effects of the above compound DH in combination with PI3K pathway inhibitors Wortmanin and LY294002 on LPS-induced NO release, survival rate and inflammatory factor expression in RAW264.7 macrophages. The specific method is as follows:

[0156] 1. Effects on NO release from RAW264.7 macrophages induced by LPS:

[0157] RAW264.7 macrophages in the logarithmic growth phase were inoculated into a 96-well plate, and 100 μL of cell suspension was added to each well. The cell density was 2.5×10 4 / mL, with 3 replicates per group, and cultured in a constant temperature incubator at 37°C and 5% CO2. After cell attachment, 300ng / mL of LPS was added to 6 groups of mouse RAW264.7 macrophages, and DMSO was used as the solvent, with DH solutions at concentrations of 0μM, 6μM and 12μM, and / or PI3K pathway inhibitors (200nM Wortmanin or 10μM LY294002) were added for co-incubation as the test drug group, where 0μM means that no compound DH was added, only 300ng / mL of LPS and PI3K pathway inhibitors were added; in addition, RAW264.7 macrophages without LPS, DH or PI3K pathway inhibitors were used as blank control groups. In addition, during the drug administration process, the PI3K pathway inhibitor was administered 1 hour in advance. After 24 hours of co-incubation, the release of cellular NO was detected.

[0158] 2. Effect on the survival rate of LPS-induced RAW264.7 macrophages:

[0159] RAW264.7 macrophages in the logarithmic growth phase were inoculated into a 96-well plate, and 100 μL of cell suspension was added to each well. The cell density was 2.5×10 4 / mL, with 3 replicates in each group, and cultured in a constant temperature incubator at 37°C and 5% CO2. After the cells adhered to the wall, 300ng / mL of LPS was added to 6 groups of mouse RAW264.7 macrophages, and different concentrations of DH solution and / or different concentrations of PI3K pathway inhibitors were added for co-incubation as the test drug group, where 0μM means that no compound DH was added, only 300ng / mL of LPS and PI3K pathway inhibitors were added; in addition, RAW264.7 macrophages without LPS, DH or PI3K pathway inhibitors were used as blank control groups. In addition, during the administration process, PI3K pathway inhibitors were administered 1 hour in advance. After 24 hours of co-incubation, 10μL of MTT reagent was added to each well, cultured at 37°C for 4 hours, DMSO was added and shaken on a shaker for 10 minutes, and the absorbance of each well was measured at 490nm using an enzyme reader to detect cell activity.

[0160] 3. Effects on the expression of inflammatory factors in RAW264.7 macrophages induced by LPS:

[0161] RAW264.7 macrophages were inoculated into 12-well plates, and 1 mL of cell suspension was added to each well. The cell density was 3×10 5 / mL, and cultured overnight in a constant temperature incubator at 37°C and 5% CO2. After cell attachment, 300ng / mL of LPS was added to 6 groups of mouse RAW264.7 macrophages, and DMSO was used as solvent, DH solution with a concentration of 6μM or 12μM, and or different concentrations of PI3K pathway inhibitors (100nM or 200nM) were added to co-incubate as the test drug group; in addition, RAW264.7 macrophages without LPS, compound DH and PI3K pathway inhibitor were used as blank control group, and mouse RAW264.7 macrophages with only LPS added were used as model group. After 12 hours of co-incubation, the mRNA expression of inflammatory factors iNOS, COX-2, IL-6, MCP-1 and CCL2 was detected by qRT-PCR.

[0162] The results are as follows Fig.19 and Fig. 20 As shown. Fig.19 It can be seen that after co-treatment of DH and wortmannin in RAW264.7 macrophages, the inhibitory effect of NO release was weakened compared with DH treatment alone. Fig. 20 As shown in the results, co-treatment with DH and LY294002 also weakened the inhibitory effect of DH alone on NO release. Further studies found that co-treatment of DH with Wortmannin or LY294002 did not eliminate the inhibitory effect of DH on the expression of pro-inflammatory factors such as iNOS, COX-2, IL-6, MCP-1 and CCL2. These results indicate that DH exerts its antioxidant and anti-inflammatory effects by activating the PI3K / AKT / mTOR signaling pathway, but the relationship is not dependent.

[0163] Experimental Example 18: Effects on LPS-induced Nrf2 / HO-1 pathway in RAW264.7 macrophages

[0164] This example detects the effect of the above compound DH on the Nrf2 / HO-1 pathway of RAW264.7 macrophages, and the specific method is as follows:

[0165] Mouse RAW264.7 macrophages were inoculated in 6-well plates, and 2000 μL of cell suspension was added to each well, with a cell density of 1×106 / mL. The cells were cultured overnight in a constant temperature incubator at 37°C and 5% CO2. After the cells adhered to the wall, 12.5 μM, 25 μM and 50 μM DH solutions and 300 μg / mL LPS were added and incubated for 24 hours as the test drug group, and RAW264.7 cells added with LPS and 40 μM commercial anti-inflammatory drug indometacin were used as the control group, and mouse RAW264.7 macrophages without LPS and DH were used as the blank control group, and mouse RAW264.7 macrophages only with LPS were used as the model group. The effect of Nrf2 nuclear translocation was observed by fluorescence microscopy, and the expression of Nrf2 and HO-1 proteins in cells was detected by Western blotting.

[0166] GAPDH was used as an internal reference during the detection process. The primary antibodies GAPDH, Nrf2, and HO-1 were purchased from Wuhan Tri-Tac (Wuhan, China), and the rabbit and mouse antibodies used as secondary antibodies were purchased from Cell Signaling Technology (Danvers, USA). The primary antibodies GAPDH and Nrf2 were diluted at a ratio of 1:5000, HO-1 was diluted at a ratio of 1:2000, and the secondary antibody was diluted at a ratio of 1:2000 before use.

[0167] Test results such as Fig.21 As shown, it was shown that compound DH significantly promoted the protein expression of Nrf2 and HO-1 in RAW264.7 macrophages. Through immunofluorescence experiments, we also directly observed the promoting effect of DH on the nuclear translocation of Nrf2, which indicated that DH could exert antioxidant effects in RAW264.7 macrophages by activating the Nrf2 / HO-1 pathway.

[0168] Experimental Example 19: Effects of the combined use of compound DH and antioxidant NAC on the Nrf2 / HO-1 pathway

[0169] This example detects the effect of the above compound DH combined with the antioxidant NAC on the Nrf2 / HO-1 pathway. For the specific experimental method, refer to the above Example 12.

[0170] Among them, DH was combined with the antioxidant NAC, NAC was administered 4 hours in advance, and the culture method was consistent with the above. After 24 hours of co-incubation, the expression levels of Nrf2 and HO-1 proteins in the cells were detected by Western blotting.

[0171] Test results such as Fig. 22As shown, the co-treatment of DH and NAC in RAW264.7 macrophages further increased the protein expression of Nrf2 and HO-1 compared with the treatment with compound DH alone, indicating that DH mainly exerts its antioxidant and anti-inflammatory effects in RAW264.7 cells by regulating oxidative stress.

[0172] Experimental Example 20: Effects on LPS-induced Nrf2 / HO-1 pathway in BMDM macrophages

[0173] The example tested the effect of the above compound DH combined with the antioxidant NAC on the Nrf2 / HO-1 pathway of BMDM macrophages induced by LPS. The specific method is as follows:

[0174] The femur and tibia of BALB / c mice were removed and the ends of the bones were trimmed. The tissue was washed with 1640 complete medium. The obtained tissue blocks were filtered through a 70μm cell sieve and the cells were recovered by centrifugation. The cells were then cultured in 1640 complete medium containing 12.5ng / mL M-CSF. On the 3rd day, fresh 1640 complete medium containing 6.25ng / mL M-CSF was replaced. On the 5th day, fresh 1640 complete medium was replaced. After 7 days of adherent culture, the bone marrow-derived precursor cells differentiated into BMDM macrophages. NAC was added to 3 groups of mouse BMDM macrophages 4 hours in advance, 800ng / mL of LPS was added to 7 groups of mouse BMDM macrophages (including the 2 groups of cells that were added with NAC 4 hours in advance), and DMSO was used as the solvent, and DH solution at concentrations of 12.5μM, 25μM, and 50μM was added for co-incubation as the test drug group; in addition, macrophages added with LPS and 40μM commercial anti-inflammatory drug indometacin were used as the control group, mouse BMDM macrophages without LPS and DH were used as the blank control group, and mouse BMDM macrophages added with only LPS were used as the model group. After 24 hours of co-incubation, the effect of Nrf2 nuclear translocation was observed by fluorescence microscopy, and the phosphorylation levels of Nrf2 and HO-1 proteins in the Nrf2 / HO-1 pathway were detected by Western blotting.

[0175] During the detection process, βaction was used as an internal reference. The primary antibodies βaction, Nrf2, and HO-1 were purchased from Wuhan Tri-Tek Co., Ltd. (Wuhan, China). The rabbit and mouse antibodies used as secondary antibodies were purchased from Cell Signaling Technology (Danvers, USA). Before use, the primary antibodies βaction and Nrf2 were diluted at a ratio of 1:5000, HO-1 was diluted at a ratio of 1:4000, and the secondary antibody was diluted at a ratio of 1:2000.

[0176] Test results such as Fig.23 As shown, it was shown that treatment with DH in BMDM macrophages can increase the expression of Nrf2 and HO-1 proteins, and the expression of Nrf2 and HO-1 proteins increased more after the combined administration of DH and NAC. In addition, immunofluorescence images also directly observed the promoting effect of DH on the nuclear translocation of Nrf2 protein in BMDM macrophages. These results are consistent with those observed in RAW264.7 cells. It confirms that DH alleviates LPS-induced oxidative stress and inflammation in both RAW264.7 and BMDM cells mainly by activating the Nrf2 / HO-1 pathway.

[0177] Experimental Example 21: Detection of the combined effect of compound DH and Nrf2 agonist

[0178] This example detected the effects of the above compound DH in combination with the Nrf2 agonist Tert-butylhydroquinone (TBHQ) on the NO release, survival rate and expression of inflammatory factors in LPS-induced RAW264.7 macrophages.

[0179] The specific method is as shown in Example 12, wherein the antioxidant NAC was replaced with the Nrf2 agonist TBHQ at a concentration of 5 μM. In addition, TBHQ was administered 3 hours in advance, and after 24 hours of co-incubation, the release of NO in RAW264.7 cells, the survival rate of RAW264.7 cells, and the mRNA expression of inflammation-related factors iNOS, IL-6, MCP-1, and CCL2 were detected.

[0180] Test results such as Fig.24 As shown, the combined use of DH and Nrf2 activator TBHQ treatment in RAW264.7 cells resulted in a further decrease in NO release compared with DH treatment alone. Compared with the LPS+DH group, the combined treatment of DH and TBHQ further attenuated the expression of inflammation-related factors iNOS, IL-6, MCP-1, and CCL2. These results indicate that DH exerts its antioxidant and anti-inflammatory effects mainly by activating Nrf2. The above experiments further confirmed that DH exerts its antioxidant and anti-inflammatory effects by regulating the production of ROS and the activation of the Nrf2 / HO-1 pathway.

[0181] In summary, the present invention provides an application of a diarylheptane compound in the preparation of an antioxidant and / or anti-inflammatory product. The above various experiments of the present invention prove that the diarylheptane compound DH separated by the present invention mainly achieves its antioxidant and anti-inflammatory effects by inhibiting the mitochondrial membrane potential to regulate the release of reactive oxygen ROS and activating the Nrf2 / HO-1 signaling pathway. And DH inhibits the expression of inflammation-related proinflammatory factors, inhibits the expression of nuclear NF-κB, inhibits the activation of the MAPK signaling pathway and the expression of iNOS and COX-2. In this process, DH also activates the PI3K / Akt / mTOR signaling pathway to produce a negative feedback effect, enhancing the inhibitory effect on the MAPK pathway and NF-κB. In addition, the compound DH of the present invention is safe and effective for cells in the concentration range of 0 to 100 μM, has no toxic side effects, and can be used to prepare antioxidants and / or anti-inflammatory drugs to eliminate oxidative stress and inflammatory reactions.

[0182] The above is a detailed description of the embodiments of the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. Use of a diarylheptane compound as shown in formula I or a pharmaceutically acceptable salt, ester, solvate, or prodrug thereof in the preparation of an antioxidant and / or anti-inflammatory product, wherein:

2. Use of the diarylheptane compound of formula I as described in claim 1 or its pharmaceutically acceptable salt, ester, solvate, prodrug in the preparation of mitochondrial membrane potential inhibitors and / or reactive oxygen species inhibitors.

3. Use of the diarylheptane compound of formula I or a pharmaceutically acceptable salt, ester, solvate or prodrug thereof as claimed in claim 1 in the preparation of an NF-κB pathway inhibitor.

4. Use of the diarylheptane compound of formula I as claimed in claim 1 or a pharmaceutically acceptable salt, ester, solvate or prodrug thereof in the preparation of a MAPK signaling pathway inhibitor.

5. Use of the diarylheptane compound of formula I or a pharmaceutically acceptable salt, ester, solvate or prodrug thereof as claimed in claim 1 in the preparation of an activator of the PI3K / Akt / mTOR signaling pathway.

6. Use of the diarylheptane compound of formula I or a pharmaceutically acceptable salt, ester, solvate or prodrug thereof as claimed in claim 1 in the preparation of an activator of the Nrf2 / HO-1 signaling pathway.

7. The use according to any one of claims 1 to 6, characterized in that: The pharmaceutically acceptable salt of the diarylheptane compound includes any one of hydrochloride, nitrate, acetate, methanesulfonate, phosphate, citrate, fumarate, sulfate, succinate, tartrate, citrate, hydrobromide, hydroiodide, lactate, toluenesulfonate, cinnamate, salicylate, malonate, glutarate and malate.

8. A pharmaceutical composition, characterized in that The active substance includes a diarylheptane compound as shown in Formula I as described in claim 1 or a pharmaceutically acceptable salt, ester, solvate, or prodrug thereof.

9. The pharmaceutical composition according to claim 8, characterized in that The pharmaceutical composition also includes pharmaceutically acceptable excipients.

10. The pharmaceutical composition according to claim 8, characterized in that The uses of the pharmaceutical composition include anti-inflammation, anti-oxidation, inhibition of mitochondrial membrane potential, inhibition of reactive oxygen species, inhibition of NF-κB pathway, inhibition of MAPK signaling pathway, activation of PI3K / Akt / mTOR signaling pathway and / or activation of Nrf2 / HO-1 signaling pathway.

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