Medicine for inhibiting nerve cell NF-kappa B inflammation signaling pathway

Abstract

The invention discloses a medicine for inhibiting a nerve cell NF-kappa B inflammation signal channel. The medicine is prepared from the following components: muscone, glycyrrhizic acid, dehydrodiisoeugenol and costunolide. The molar concentrations of the musk ketone, the glycyrrhizic acid, the dehydrodiisoeugenol and the costunolide are all 0.1 to 0.2 nM. The medicine for inhibiting the nerve cell NF-kappa B inflammation signal channel has the advantages that the nerve cell NF-kappa B inflammation signal channel can be inhibited on the nanomolar concentration level, and the treatment effect can still be achieved after the administration amount and the administration frequency are reduced.

Description

Technical Field

[0001] The present invention relates to the field of anti-inflammatory drugs, and in particular to a drug for inhibiting the NF-κB inflammatory signal pathway of nerve cells. Background Art

[0002] The NF-κB inflammatory signaling pathway of microglia (BV2 cells) can be found in almost all animal cells. They are involved in the response of cells to external stimuli, such as cytokines, radiation, heavy metals, viruses, etc. NF-κB plays a key role in the process of cell inflammation and immune response. Misregulation of NF-κB can cause 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 studies have shown that muscone can significantly inhibit the NF-κB inflammatory signaling pathway at a molar concentration of 2 µM, and can significantly inhibit the NF-κB inflammatory signaling pathway at 25.16 µM (6 µg / mL). Glycyrrhizic acid can inhibit the NF-κB inflammatory signaling pathway at a molar concentration of 10 µM. Dehydrodiisoeugenol can inhibit the NF-κB inflammatory signaling pathway at a molar concentration of 1 µM. Costunolide ((+)-costunolide, costunolide) can inhibit the NF-κB inflammatory signaling pathway at a molar concentration of 2.5 µM. These drugs can also inhibit inflammatory responses on other cells, but they can only inhibit inflammation at a concentration level of micromoles per liter, and their effective concentration levels are relatively high. Moreover, these natural products exhibit limitations such as low hydrophilicity, low bioavailability or poor permeability. Their effects when applied in vivo are not ideal and require repeated administration outside the safe range, which is not conducive to the long-term treatment of chronic diseases. Summary of the invention

[0004] In order to solve the above technical problems, the purpose of the present invention is to provide a drug for inhibiting the NF-κB inflammatory signaling pathway of neurons, so as to inhibit the NF-κB inflammatory signaling pathway of neurons 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 scheme: a drug for inhibiting the NF-κB inflammatory signaling pathway of nerve cells, the drug components including musk ketone, glycyrrhizic acid, dehydrogenated diisoeugenol, and costunolide; the molar concentrations of the musk ketone, the glycyrrhizic acid, the dehydrogenated diisoeugenol, and the costunolide are all 0.1-0.2 nM.

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

[0007] Advantages of the present invention: The drug for inhibiting the NF-κB inflammatory signaling pathway of nerve cells provided by the present invention can inhibit the NF-κB inflammatory signaling pathway of 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 embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0009] Figure 1 The figure is a diagram showing the inhibitory effect of Example 1 of the present invention as the experimental group and the control group on the NF-κB inflammatory signaling pathway.

[0010] Figure 2 This is a diagram showing the inhibitory effect of the experimental group and the control group on the NF-κB inflammatory signaling pathway in Example 2 of the present invention. DETAILED DESCRIPTION

[0011] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0012] Example 1: A drug for inhibiting the NF-κB inflammatory signaling pathway of nerve cells, the drug components include musk ketone, glycyrrhizic acid, dehydrodiisoeugenol, and costunolide; the molar concentration of musk ketone, glycyrrhizic acid, dehydrodiisoeugenol, and costunolide is 0.1 nM. The 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 of nerve cells, the drug components include musk ketone, glycyrrhizic acid, dehydrodiisoeugenol, and costunolide; the molar concentration of musk ketone, glycyrrhizic acid, dehydrodiisoeugenol, and costunolide is 0.2 nM. The drug can significantly inhibit the activation of the NF-κB inflammatory signaling pathway induced by LPS in BV2 nerve cells.

[0014] Embodiment 3: Experimental methods: (1) musk ketone, glycyrrhizic acid, dehydrodiisoeugenol, and costunolide are mixed and temporarily stored as the MGDC group, wherein the mixture is mixed according to the ratio in Example 1, which is recorded as MIX1; the mixture is mixed according to the ratio in Example 2, which is recorded as MIX2; and the mixture is mixed according to the molar concentration of musk ketone, glycyrrhizic acid, dehydrodiisoeugenol, and costunolide at 1 nM, which is recorded as MIX3; (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 amounts of Opti-MEM medium (Thermo); (3) Mix the diluted lipofectamine 2000 transfection reagent with DNA and incubate at room temperature for 20 minutes; (4) Add the mixture in step (3) to BV2 cells and incubate at 37°C for 6 hours; (5) 48 hours after transfection of BV2 microglia; (6) Treat the BV2 microglial cells from the previous step with different concentrations of drugs and 1 µg / mL LPS overnight; (7) Luciferase activity of the NF-κB reporter gene was detected using a luciferase reporter gene assay kit (Promega, Madison, USA). The NF-κB luciferase reporter plasmid pGL4.32 [luc2P NF-kB-RE Hygro] contains five copies of the NF-κB response element (NF-κB-RE). 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 is detected by the luciferase reporter gene assay kit. Three wells were prepared on a 12-well plate for the blank group, LPS stimulation group, and different concentrations of drug administration groups. After induction, chemiluminescence detection was performed. Cells were collected first, and then 300 µl of chemiluminescence reagent was added to each well. After resuspending the cells, 100 µl was added to a 96-well plate. The fluorescence intensity was detected by chemiluminescence mode, and the detection time for each well was 1000 ms on a multifunctional microplate reader (Infinite 200 PRO, TECAN, Austria); (8) Calculate the data according to the following formula to obtain the NF-κB (relative luciferase activity) of each group of experiments: NF-κB transcriptional activity = relative light units of firefly luciferase / relative light units of Renilla luciferase NF-κB (relative luciferase activity) = NF-κB transcription activity / blank group NF-κB transcription activity * 100 (9) Graph Pad Prism (version 6.02) was used for statistical analysis. One-way ANOVA and Dunnett's multiple comparison tests were used for significant differences between the LPS-induced group and other groups (* p <0.05, ** p <0.01, ns= p >0.05).

[0015] The NF-κB inflammatory signaling pathway without any drug was used as a blank group, LPS was used as a positive control group for activating the NF-κB inflammatory signaling pathway, and then the NF-κB inflammatory signaling pathway of BV2 neurons induced by LPS (final concentration of 500 ng / ml) was treated with musk ketone at molar concentrations of 0.1 nM and 1 nM, glycyrrhizic acid at molar concentrations of 0.1 nM and 1 nM, dehydrodiisoeugenol at molar concentrations of 0.1 nM and 1 nM, and costunolide at molar concentrations of 0.1 nM and 1 nM, respectively. The results are shown in Tables 1 and 2. The MGDC experimental group MIX1 was treated with drugs according to Example 1, and the MGDC experimental group MIX3 was treated according to the drugs according to Example 1. Figure 1 As shown; the NF-κB inflammatory signaling pathway without any drug was used as the blank group, LPS was used as the positive control group for activating the NF-κB inflammatory signaling pathway, and then the NF-κB inflammatory signaling pathway of BV2 neurons induced by LPS (final concentration 500 ng / ml) was treated with 0.2 nM musk ketone, 0.2 nM glycyrrhizic acid, 0.2 nM dehydrodiisoeugenol, and 0.2 nM costusinol as a single component control group, and the MGDC experimental group MIX2 was treated with drugs according to Example 2. The results are shown in FIG. Figure 2 shown.

[0016] Table 1 NF-κB (relative luciferase activity) detected and calculated after each experimental treatment

[0017] Results analysis: From Table 1 and Figure 1It can be seen that the musk ketone group (0.1 nM and 1 nM), glycyrrhizic acid group (0.1 nM and 1 nM), dehydrodiisoeugenol group (0.1 nM and 1 nM), and costunolide group (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) at 0.1 nM could significantly inhibit the NF-κB inflammatory signaling pathway of BV2 neurons induced by LPS, which was 33.2% higher than the musk ketone group (1 nM) alone, 32.2% higher than the glycyrrhizic acid group (1 nM) alone, 34.1% higher than the dehydrodiisoeugenol group (1 nM) alone, and 36.6% higher than the costunolide group (1 nM) alone. Figure 2 It can be seen that the drug components include musk ketone, glycyrrhizic acid, dehydrodiisoeugenol, and costusinol; when the molar concentrations of musk ketone, glycyrrhizic acid, dehydrodiisoeugenol, and costusinolol are all 0.2 nM, the inhibitory effect is most significant.

[0018] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A drug for inhibiting the NF-κB inflammatory signaling pathway of nerve cells, characterized in that: The drug components include musk ketone, glycyrrhizic acid, dehydrogenated diisoeugenol and costunolide; the molar concentrations of the musk ketone, glycyrrhizic acid, dehydrogenated diisoeugenol and costunolide are all 0.1-0.2 nM.

2. A drug for inhibiting the NF-κB inflammatory signaling pathway of nerve cells according to claim 1, characterized in that: The molar concentration ratio of the musk ketone, the glycyrrhizic acid, the dehydrodiisoeugenol and the costunolide is 1:1:1:1.

Images