A drug for inhibiting the NF-κB inflammatory signaling pathway in nerve cells

Abstract

The present invention discloses a drug for inhibiting the NF-κB inflammatory signaling pathway in nerve cells. The drug components include muskone, glycyrrhizic acid, dehydrodiisoeugenol, and costunolide; the molar concentrations of the muskone, the glycyrrhizic acid, the dehydrodiisoeugenol, and the costunolide are all 0.1-0.2 nM. Advantage: The drug for inhibiting the NF-κB inflammatory signaling pathway in nerve cells provided by the present invention can inhibit the NF-κB inflammatory signaling pathway in nerve cells at the nanomolar concentration level, so that the therapeutic effect can still be achieved after reducing the dosage and the number of administrations.

Description

Technical Field

[0001] The present invention relates to the field of anti-inflammatory drugs, and particularly to a drug that inhibits the NF-κB inflammatory signaling pathway in nerve cells. Background Art

[0002] The NF-κB inflammatory signaling pathway can be found in almost all animal cells in microglial cells (BV2 cells), which are involved in the cell's response to external stimuli, such as cytokines, radiation, heavy metals, viruses, etc.; NF-κB plays a key role in processes such as the cell's inflammatory response and immune response; and the misregulation of NF-κB can trigger autoimmune diseases, chronic inflammation, and many cancers.

[0003] Studies have found that lipopolysaccharide (LPS) can induce the NF-κB inflammatory signaling pathway in microglial cells (BV2 cells), and there are studies showing that when the molar concentration of muscone is 2 μM, it can significantly inhibit the NF-κB inflammatory signaling pathway, and at 25.16 μM (6 μg / mL), it can significantly inhibit the NF-κB inflammatory signaling pathway. When the molar concentration of glycyrrhizic acid is 10 μM, it can inhibit the NF-κB inflammatory signaling pathway. When the molar concentration of dehydrodiisoeugenol is 1 μM, it can inhibit the NF-κB inflammatory signaling pathway. When the molar concentration of (+)-costunolide (costunolide) is 2.5 μM, it can inhibit the NF-κB inflammatory signaling pathway. These drugs can also inhibit the inflammatory response in other cells, but they can only achieve the effect of inhibiting inflammation at the concentration level of micromoles per liter, and the concentration level at which they take effect is relatively high. Moreover, these natural products exhibit limitations such as low hydrophilicity, low bioavailability, or poor permeability, and the effects when applied in the body are not ideal, and repeated administration outside the safe range is required, which is not conducive to the long-term treatment of chronic diseases. Summary of the Invention

[0004] To solve the above technical problems, the purpose of the present invention is to provide a drug that inhibits the NF-κB inflammatory signaling pathway in nerve cells, so as to achieve the inhibition of the NF-κB inflammatory signaling pathway in nerve cells at the nanomolar concentration level, so that the therapeutic effect can still be achieved after reducing the dosage and the number of administrations.

[0005] The purpose of the present invention is implemented by the following technical solution: A drug that inhibits the NF-κB inflammatory signaling pathway in nerve cells, the drug components include muscone, glycyrrhizic acid, dehydrodiisoeugenol, and (+)-costunolide; the molar concentrations of the muscone, the glycyrrhizic acid, the dehydrodiisoeugenol, and the (+)-costunolide are all 0.1 - 0.2 nM.

[0006] Furthermore, the molar concentration ratio of the muskone, glycyrrhizic acid, dehydrodiisoeugenol, and costunolide is: 1:1:1:1.

[0007] Advantages of the present invention: The drug provided by the present invention for inhibiting the NF-κB inflammatory signaling pathway in nerve cells can inhibit the NF-κB inflammatory signaling pathway in nerve cells at the nanomolar concentration level, so that the therapeutic effect can still be achieved after reducing the dosage and the number of administrations. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0009] Figure 1 It is the inhibitory effect diagram of the NF-κB inflammatory signaling pathway with Example 1 of the present invention as the experimental group and the control group.

[0010] Figure 2 It is the inhibitory effect diagram of the NF-κB inflammatory signaling pathway with Example 2 of the present invention as the experimental group and the control group. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0011] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the drawings in the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0012] Example 1: A drug for inhibiting the NF-κB inflammatory signaling pathway in nerve cells, the drug components include muskone, glycyrrhizic acid, dehydrodiisoeugenol, and costunolide; the molar concentrations of muskone, glycyrrhizic acid, dehydrodiisoeugenol, and costunolide are all 0.1 nM. This drug can significantly inhibit the activation of the NF-κB inflammatory signaling pathway induced by LPS in BV2 nerve cells.

[0013] Example 2: A drug for inhibiting the inflammatory pathway in nerve cells, the drug components include muskone, glycyrrhizic acid, dehydrodiisoeugenol, and costunolide; the molar concentrations of muskone, glycyrrhizic acid, dehydrodiisoeugenol, and costunolide are all 0.2 nM. This drug can significantly inhibit the activation of the NF-κB inflammatory signaling pathway induced by LPS in BV2 nerve cells.

[0014] Example 3:

[0015] Experimental method:

[0016] (1) Mix muskone, glycyrrhizic acid, dehydrodiisoeugenol, and costunolide as the MGDC group for temporary storage. Among them, mix according to the ratio of Example 1, denoted as MIX1; mix according to the ratio of Example 2, denoted as MIX2; mix with the molar concentrations of muskone, glycyrrhizic acid, dehydrodiisoeugenol, and costunolide all being 1 nM, denoted as MIX3;

[0017] (2) Dilute Lipofectamine 2000 transfection reagent (Thermo, Waltham, MA, USA) and DNA (pGL4.32 plasmid, manufacturer: Promega, catalog number: E8491, NF-κB plasmid with firefly luciferase) into equal volumes of Opti-MEM medium (Thermo);

[0018] (3) Mix the diluted lipofectamine 2000 transfection reagent with DNA and then incubate at room temperature for 20 minutes;

[0019] (4) Add the mixture in step (3) to BV2 cells and incubate at 37°C for 6 hours;

[0020] (5) 48 hours after transfecting BV2 microglial cells;

[0021] (6) Treat the BV2 microglial cells in the previous step with drugs at different concentrations and 1 μg / mL LPS overnight;

[0022] (7) The luciferase reporter gene assay kit (Promega, Madison, USA) was used to detect the luciferase activity of the NF-κB reporter gene (NF-κB luciferase reporter plasmid pGL4.32 [luc2P NF-kB-RE Hygro], the plasmid contains five copies of the NF-κB response element (NF-κB-RE), and the activation of this element (such as the active inducer LPS) drives the transcription of the luciferase reporter gene luc2P (Photinus pyralis). The activity of the luciferase reporter gene luc2P was detected by the luciferase reporter gene assay kit). In the 12-well plate, three wells were set up for the blank group, the LPS-stimulated group, and the drug-administered groups at different concentrations. After induction, chemiluminescence detection was performed. First, the cells were collected, then 300 µl of chemiluminescence reagent was added to each well. After resuspending the cells, 100 µl was taken and added to the 96-well plate. The fluorescence intensity was detected in the chemiluminescence mode, and the detection time for each well was 1000 ms. The analysis was carried out on a multifunctional microplate reader (Infinite 200 PRO, TECAN, Austria);

[0023] (8) The data was calculated according to the following formula to obtain the NF-κB (relative luciferase activity) of each group of experiments:

[0024] NF-κB transcriptional activity = relative light units of firefly luciferase / relative light units of Renilla luciferase

[0025] NF-κB (relative luciferase activity) = NF-κB transcriptional activity / NF-κB transcriptional activity of the blank group * 100

[0026] (9) Statistical analysis was performed using the Graph Pad Prism (version 6.02) method. One-way ANOVA was used for significance comparison, and Dunnett's multiple comparison tests were used for the differences between the LPS-induced group and other groups (* p <0.05, ** p <0.01, ns = p >0.05).

[0027] The NF-κB inflammatory signaling pathway without any drug added was used as the blank group, and LPS was used as the positive control group to activate the NF-κB inflammatory signaling pathway. Then, the NF-κB inflammatory signaling pathway of BV2 neurons induced by LPS (final concentration 500 ng / ml) was treated with muscone at molar concentrations of 0.1 nM and 1 nM, glycyrrhizic acid at 0.1 nM and 1 nM, dehydrodiisoeugenol at 0.1 nM and 1 nM, and costunolide at 0.1 nM and 1 nM as single-component control groups, and treated according to the drug in Example 1 as the MGDC experimental group MIX1, and treated according to the MGDC experimental group MIX3. The results are shown in Table 1, Figure 1 as follows; the NF-κB inflammatory signaling pathway without any drug added was used as the blank group, and LPS was used as the positive control group to activate the NF-κB inflammatory signaling pathway. Then, the NF-κB inflammatory signaling pathway of BV2 neurons induced by LPS (final concentration 500 ng / ml) was treated with muscone at a molar concentration of 0.2 nM, glycyrrhizic acid at 0.2 nM, dehydrodiisoeugenol at 0.2 nM, and costunolide at 0.2 nM as single-component control groups, and treated according to the drug in Example 2 as the MGDC experimental group MIX2. The results are as Figure 2 shown.

[0028] Table 1 Table of NF-κB (relative luciferase activity) obtained by detection and calculation after experimental treatments of each group

[0029]

[0030] Result analysis: From Table 1 and Figure 1 it can be seen that the muscone groups (0.1 nM and 1 nM), glycyrrhizic acid groups (0.1 nM and 1 nM), dehydrodiisoeugenol groups (0.1 nM and 1 nM), and costunolide groups (0.1 nM and 1 nM) could not significantly inhibit the NF-κB inflammatory signaling pathway of BV2 neurons induced by LPS. The MGDC group (MIX1) could significantly inhibit the NF-κB inflammatory signaling pathway of BV2 neurons induced by LPS at 0.1 nM. Compared with the effect of the muscone group (1 nM) alone in inhibiting the NF-κB inflammatory signaling pathway, it was increased by 33.2%. Compared with the effect of the glycyrrhizic acid group (1 nM) alone in inhibiting the NF-κB inflammatory signaling pathway, it was increased by 32.2%. Compared with the effect of the dehydrodiisoeugenol group (1 nM) alone in inhibiting the NF-κB inflammatory signaling pathway, it was increased by 34.1%. Compared with the effect of the costunolide group (1 nM) alone in inhibiting the NF-κB inflammatory signaling pathway, it was increased by 36.6%. From Figure 2It can be known that the drug components include muscone, glycyrrhizic acid, dehydrodiisoeugenol, and costunolide; when the molar concentrations of muscone, glycyrrhizic acid, dehydrodiisoeugenol, and costunolide are all 0.2 nM, the inhibitory effect is the most significant.

[0031] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A drug for inhibiting the NF-κB inflammatory signaling pathway in nerve cells, characterized in that, The pharmaceutical components include muscone, glycyrrhizic acid, dehydrodiisoeugenol, and costunolide; the molar concentrations of the muscone, the glycyrrhizic acid, the dehydrodiisoeugenol, and the costunolide are all 0.1 - 0.2 nM; The molar concentration ratio of the muscone, the glycyrrhizic acid, the dehydrodiisoeugenol, and the costunolide is: 1:1:1:1.

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