Application of retinaldehyde in preparation of medicine for treating Toll-like receptor mediated inflammation and medicine

Abstract

The invention belongs to the field of new application of medicines, and particularly relates to application of retinaldehyde in preparation of medicines for treating Toll-like receptor mediated inflammation and the medicines. It is found for the first time that retinaldehyde can be used for inhibiting TLR-mediated inflammatory reaction through targeted activation of OPN3, experimental results show that after all-type retinaldehyde and 9-cis-retinaldehyde are treated, the expression quantity of TNF, IL6, IL18, IFNB1 and ISG15 inflammatory genes in human mononuclear macrophages stimulated by PAM3CSK4, VTX-2337, TNF, Agatolimod and Poly (I: C) can be remarkably reduced, transcriptome experimental data show that the expression quantity of TNF, IL6, IL18, IFNB1 and ISG15 inflammatory genes in the human mononuclear macrophages can be remarkably reduced, and the expression quantity of TNF, IL6, IL18, IFNB1 and ISG15 inflammatory genes can be remarkably The all-form retinaldehyde and the 9-cis-retinaldehyde can activate OPN3 and are used for inhibiting TLR-mediated inflammatory reaction, so that a new thought is provided for treatment of inflammation, and the pharmaceutical application of the retinaldehyde is expanded.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of new uses of drugs, and more particularly relates to the use of retinal in the preparation of a drug for treating Toll-like receptor-mediated inflammation. BACKGROUND

[0002] Inflammation-related diseases have seriously affected human life and health and quality of life for a long time, and strict regulation of inflammatory response and maintenance of immune balance are crucial for maintaining homeostasis of the body.

[0003] Opsins (OPNs) are a family of light-sensitive G protein-coupled receptors with 7 transmembrane domains, and their main role is to bind retinal as a binding site using the lysine residue of the seventh helix to participate in multidimensional light transmission. OPN3 is a member of the OPN family, also known as encephalopsin or panopsin, and was first discovered in the brain tissue of mice in 1999 and is mainly expressed in organs such as the eye, brain and liver. Although OPN3 has been discovered for more than 20 years, its functional characteristics and expression profile are still not very clear. Studies have shown that OPN3 and its homologues can form functional photopigments with 11-cis-retinal, 9-cis-retinal and all-trans retinal (atRal). Mammalian OPN3 is different from its homologues in insects and fish in terms of phylogeny, and therefore does not have the same light-sensitive effect as insects and fish. It has been reported that OPN3 is expressed on adipocytes, participates in adipocyte metabolism, and can inhibit obesity and insulin resistance induced by a high-fat diet in mice. Retinal is a derivative of vitamin A (retinoids), and it has also been reported that retinoids in lung tissue can reduce LPS-induced acute lung injury. However, the specific mechanism by which retinoids inhibit LPS-induced inflammation and lung injury, and whether OPN3 mediated by retinal is involved, is still unclear.

[0004] Toll-like receptors (TLRs) are important protein molecules involved in non-specific immunity, and TLR3 / 7 / 8 / 9 are expressed on endosomes and are important receptors for recognizing DNA and RNA of bacterial and viral origin. Activation of these TLRs induces the release of IFN-I and inflammatory factors, which can cause acute lung inflammation and lung tissue damage. Therefore, inhibition of TLR-mediated inflammatory response will help to reduce tissue inflammation and damage and improve the survival rate of the body.

[0005] Retinoids, including 9-cis-retinal and all-trans-retinal, are important signal transduction molecules in eye development, which have a variety of different important roles in eye development of vertebrates, and the current research on retinoids mainly focuses on visual physiology research, retinal disease mechanism and treatment research. In order to further expand the application range of retinoids, further research on other pharmaceutical uses of retinoids is still needed. SUMMARY

[0006] The application aims to provide the application of retinoids in the preparation of drugs for treating Toll-like receptor mediated inflammation, so as to solve the above technical problems.

[0007] In order to achieve the above-mentioned purpose, the technical scheme adopted by the application is as follows: The application of retinoids in the preparation of drugs for treating Toll-like receptor mediated inflammation.

[0008] Retinoids are active aldehyde forms of vitamin A (retinol), which are composed of a beta-ionone ring and a polyene chain, and exist in photoreceptor cells (rods and cones) of the retina, and are a component of rhodopsin. The application first finds that retinoids can be used to inhibit TLR mediated inflammatory response by targeting activation of OPN3, which can significantly reduce the expression amount of TNF, IL6, IL18, IFNB1 and ISG15 inflammatory genes in human monocyte macrophages, and provides a new idea for the treatment of inflammation, and expands the pharmaceutical use of retinoids.

[0009] Further, the retinoids are used as the only effective component in the drug.

[0010] Further, the drug further comprises a pharmaceutically acceptable excipient.

[0011] Further, the excipient comprises any one or more of a filler, a stabilizer, a diluent, and an adjuvant.

[0012] Further, the diluent is any one of water and normal saline.

[0013] Based on the same inventive concept, the application further provides a drug for treating Toll-like receptor mediated inflammation, which is prepared by mixing the retinoids and an excipient, and the content of the retinoids in the drug is 0.1wt%-99wt%.

[0014] Further, the dosage form of the drug is a solid dosage form or a solution dosage form, the solid dosage form comprises granules, tablets, capsules, pills, and dripping pills, and the solution dosage form comprises oral liquid preparations, intragastric administration preparations, and injection administration dosage forms.

[0015] Further, the solution dosage form is a solution of water and the retinol, or a solution of physiological saline and the retinol.

[0016] Advantages: The present application has found that retinol can significantly reduce the expression of TNF, IL6, IL18, IFNB1 and ISG15 inflammatory genes caused by PAM3CSK4, VTX-2337, TNF, Agatolimod and Poly(I:C) stimulation by targeting the activation of OPN3 for inhibiting TLR-mediated inflammatory response, which is of great significance for reducing tissue inflammation and damage caused by inflammation and improving the survival rate of the body. This provides a new candidate drug for clinical treatment of inflammatory damage. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 Figure is a graph of OPN3 expression.

[0018] Figure 2 Figure is an inflammatory gene expression graph after PAM3CSK4 treatment, wherein A is a TNF gene expression statistical graph, B is an IL6 gene expression statistical graph, C is an IL18 gene expression statistical graph, D is an IFNB1 gene expression statistical graph, and D is an ISG15 gene expression statistical graph.

[0019] Figure 3 Figure is an inflammatory gene expression graph after VTX-2377 treatment, wherein A is a TNF gene expression statistical graph, B is an IL6 gene expression statistical graph, C is an IL18 gene expression statistical graph, D is an IFNB1 gene expression statistical graph, and D is an ISG15 gene expression statistical graph.

[0020] Figure 4 Figure is an inflammatory gene expression graph after Agatolimod treatment, wherein A is a TNF gene expression statistical graph, B is an IL6 gene expression statistical graph, C is an IL18 gene expression statistical graph, D is an IFNB1 gene expression statistical graph, and D is an ISG15 gene expression statistical graph.

[0021] Figure 5 Figure is an inflammatory gene expression graph after Poly(I:C) treatment, wherein A is a TNF gene expression statistical graph, B is an IL6 gene expression statistical graph, C is an IL18 gene expression statistical graph, D is an IFNB1 gene expression statistical graph, and D is an ISG15 gene expression statistical graph.

[0022] Figure 6Figure 9 is a graph of TLR8 ligand-mediated inflammatory gene expression inhibition by 9-cis-RAL, wherein A is a graph of TNF gene expression, B is a graph of IL6 gene expression, C is a graph of IL18 gene expression, D is a graph of IFNB1 gene expression, and D is a graph of ISG15 gene expression.

[0023] Figure 7 Figure 10 is a principal component analysis graph.

[0024] Figure 8 Figure 11 is a K-means clustering heat map of differential genes.

[0025] Figure 9 Figure 12 is a heat map of inflammatory gene expression.

[0026] Figure 10 Figure 13 is a graph of inflammatory genes (Inflammatory genes) and immediate early genes (IEGs) expression. DETAILED DESCRIPTION

[0027] The present application will be described in detail below with reference to the accompanying drawings and specific examples, but should not be understood as limiting the present application. If not specifically stated, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, if not specifically stated, can be obtained from commercial channels.

[0028] Example 1 I. Experimental materials

[0029] All-trans-retinal (atRAL), 9-cis-retinal (9-cis-RAL), PAM3CSK4, VTX-2337, TNF, Agatolimod, and Poly(I:C) were purchased from MCE. LPS was purchased from Sigma. Reverse transcription kit and real-time fluorescent PCR kit were provided by Novozyme. CD14 sorting kit was purchased from Meitenyi. Sorting column was provided by Shenzhen Ruivode Life Science and Technology Co., Ltd. Other consumables were purchased from Shanghai Huiwang Industry Co., Ltd.

[0030] II. Experimental methods

[0031] 1、Human monocyte macrophage: collect peripheral blood into 50 mL centrifuge tube, dilute with PBS to 30 mL, mix well after blowing, slowly pour into the surface of 8 mL lymphocyte separation medium, centrifuge at room temperature 2000 rpm / min for 20 min, after centrifugation, the mononuclear cell layer is aspirated into a new 50 mL centrifuge tube, add PBS to 30 mL, centrifuge at room temperature 1600 rpm / min for 10 min, resuspend the cells with PBS, and add CD14 sorting magnetic beads for incubation at 4℃ for 20 min. Then put the sorting column on the magnetic stand, add 3 mL PBS, wait for the sorting column to stop dripping, then add the incubated cell suspension, stop dripping, then wash with 3 mL PBS for 3 times, then wash out the CD14 sorting magnetic bead labeled cells with 5 mL PBS, repeat the operation once, then count the cells and perform subsequent experiments.

[0032] 2、Cell culture and grouping experiment: human monocyte macrophages are cultured in 1640 medium containing 10% fetal bovine serum, 50 μg / mL penicillin and 50 µg / mL streptomycin. The culture is placed in a 37℃, 5% carbon dioxide incubator. Different TLR agonists are treated in groups, and after 3h incubation with all-trans retinol or 9-cis-retinal, the cells are collected for real-time quantitative polymerase chain reaction and transcriptome sequencing.

[0033] III. Sample detection.

[0034] 1、RNA extraction and real-time fluorescence quantitative polymerase chain reaction: add 200 μL pre-cooled Trizol reagent to an equal amount of cells, add 40 μL chloroform after 5 min of lysis, mix well by shaking for 30 s, then stand for 3 min, then centrifuge at 12000 rpm / min, 4℃ for 15 min. Transfer the supernatant to a new EP tube and mix with an equal amount of isopropanol, stand at room temperature for 10 min, then centrifuge at 12000 rpm / min for 10 min, discard the supernatant. Add 1 ml of 75% ethanol, then centrifuge the supernatant at 7500 rpm / min for 5 min. Discard the supernatant and dry the precipitate. Use the Novozyme reverse transcription kit to detect the mRNA levels of TNF-α, IL-6, IL18, IFNB1, ISG15, IFIT1, etc. GAPDH gene expression is normalized. The primer sequences are shown in the following table.

[0035] Table 1: Primer sequences 2. Transcriptome sequencing: Human monocyte macrophages were cultured in 1640 medium containing 10% fetal bovine serum, 50 μg / mL penicillin and 50 μg / mL streptomycin. Ctrl group treatment was DMSO (20 μM) incubation to stimulate human monocyte macrophages for 3 h, VTX group treatment was VTX-2377 (10 μM) to stimulate human monocyte macrophages for 3 h, atRAL group was atRAL (20 μM) to stimulate human monocyte macrophages for 3 h, atRAL + VTX group treatment was VTX (10 μM) and atRAL (20 μM) used in combination to stimulate human monocyte macrophages for 3 h, and samples were collected after 3 h. The samples were sent to Bioinformatics Technology Co., Ltd. for mRNA transcriptome sequencing.

[0036] Four, experimental data analysis.

[0037] 1. Statistical analysis: All data were expressed as mean ± standard deviation. One-way ANOVA was used for analysis, and T test was used for experimental data by GraphPad Prism software and graphical representation. P value < 0.05, the difference was statistically significant. Mean ± SEM, n = 3, ns-not significance. * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, **** represents p < 0.0001.

[0038] 2. Transcriptome data analysis: The results of transcriptome data analysis were provided by Bioinformatics Technology Co., Ltd.

[0039] Five, experimental results.

[0040] 1. OPN3 expression increased under the induction of TLR ligand: The experimental results are shown in Figure 1 , Ctrl group is human monocyte macrophages cultured in 1640 complete medium, Pam3 group treatment is to stimulate human monocyte macrophages cultured in 1640 complete medium with TLR2 ligand PAM3CSK4 at a concentration of 1 μg / ml for 3 h, LPS group treatment is to stimulate human monocyte macrophages cultured in 1640 complete medium with TLR4 ligand LPS at a concentration of 10 ng / ml for 3 h, VTX group treatment is to stimulate human monocyte macrophages cultured in 1640 complete medium with TLR8 ligand VTX-2337 at a concentration of 10 μM for 3 h, TNF group treatment is to stimulate human monocyte macrophages cultured in 1640 complete medium with TNF at a concentration of 20 ng / ml for 3 h, and the expression of OPN3 was detected after stimulation. The experimental results show that the expression of OPN3 gene increases under the induction of PAM3CSK4, LPS and VTX-2337.

[0041] 2, atRal and 9-cis-RAL activated OPN3 inhibited TLR-mediated inflammatory response: The experimental results are shown in Figure 2 Figure 6A, the Ctrl group was incubated with 20 mM atRal and 20 mM DMSO, respectively, to stimulate human monocyte macrophages for 3 hours, the Pam3 group was treated with 1 pg / ml PAM3CSK4 and 20 mM atRal or 20 mM DMSO, respectively, to stimulate human monocyte macrophages for 3 hours, the LPS group was treated with 10 ng / ml LPS and 20 mM atRal or 20 mM DMSO, respectively, to stimulate human monocyte macrophages for 3 hours, and the results showed that the expression of TNF, IL6, IFNB1 and ISG15 genes induced by PAM3CSK4 and LPS was reduced.

[0042] The experimental results are shown in Figure 3 Figure 6B, the Agatolimod group was treated with 10 pg / ml Agatolimod and 20 mM atRal or 20 mM DMSO, respectively, to stimulate human monocyte macrophages for 3 hours, and the results showed that the expression of inflammatory genes induced by Agatolimod was reduced.

[0043] The experimental results are shown in Figure 4 Figure 6C, the VTX group was treated with 10 mM VTX-2377 and 20 mM atRal or 20 mM DMSO, respectively, to stimulate human monocyte macrophages for 3 hours, and the results showed that the expression of inflammatory genes induced by VTX-2377 was reduced.

[0044] The experimental results are shown in Figure 5 Figure 6D, the TLR3 ligand Poly(I:C) was treated with 25 pg / ml Poly(I:C) and 20 mM atRal or 20 mM DMSO, respectively, to stimulate human monocyte macrophages for 3 hours, and the results showed that the expression of inflammatory genes induced by Poly(I:C) was reduced.

[0045] The experimental results are shown in Figure 6 Figure 6E, the VTX group was treated with 10 mM VTX-2377 and 20 mM 9-cis-RAL or 20 mM DMSO, respectively, to stimulate human monocyte macrophages for 3 hours, and the results showed that the expression of inflammatory genes induced by VTX-2377 was reduced.

[0046] The experimental results are shown in Figure 7 Figure 6F, principal component analysis of mRNA transcriptome data found that the Ctrl group, the VTX group, the atRal group and the atRal+VTX group were obviously separated. Figure 8For the four groups of post-treatment differential genes more than two times of K-means clustering (K=7), the false discovery rate (FDR) is less than 0.05, the results show that compared with VTX-2377 treatment alone, the gene expression has significant difference after adding atRAL.

[0047] The experimental results are shown in Figure 9 RAL can reduce the expression of inflammation genes induced by VTX-2377. Figure 10 For the heat map of inflammation gene expression and rapid response gene (IEG), the experimental results show that adding atRAL reduces the expression of inflammation genes and immediate early genes (IEGs) induced by VTX-2377. These results show that atRal and 9-cis-RAL activating OPN3 can be used to inhibit TLR-mediated inflammatory response. Therefore, the present application finds that retinaldehyde targeting OPN3 can be used to inhibit TLR-mediated inflammatory response.

[0048] It should be noted that when the present application claims involve numerical ranges, it should be understood that each numerical range of two endpoints and any numerical value between the two endpoints can be selected. In order to prevent repetition, the present application describes the preferred embodiments.

[0049] Although the preferred embodiments of the present application have been described, those skilled in the art can make further changes and modifications to these embodiments once they know the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications falling within the scope of the present application.

[0050] Obviously, those skilled in the art can make various modifications and variations to the present application without departing from the spirit and scope of the present application. Thus, if these modifications and variations of the present application fall within the scope of the claims of the present application and their equivalents, the present application also intends to include these modifications and variations.

Claims

1. Use of retinene in the manufacture of a medicament for treating Toll-like receptor mediated inflammation.

2. Use of retinol for the preparation of a medicament for the treatment of Toll-like receptor mediated inflammation according to claim 1, characterized in that, The medicament comprises retinene as the only effective component.

3. Use of retinol for the preparation of a medicament for the treatment of Toll-like receptor mediated inflammation according to claim 1, characterized in that, The medicament further comprises pharmaceutically acceptable adjuvants.

4. Use of retinol for the preparation of a medicament for the treatment of Toll-like receptor mediated inflammation according to claim 3, characterized in that, The adjuvants comprise any one or more of fillers, stabilizers, diluents, adjuvants.

5. Use of retinol for the preparation of a medicament for the treatment of Toll-like receptor mediated inflammation according to claim 4, characterized in that, The diluents are any one of water and normal saline.

6. A medicament for treating Toll-like receptor-mediated inflammation, characterized by, The medicament is prepared by mixing the retinene and the adjuvants as described in claim 1, and the content of the retinene in the medicament is 0.1wt% to 99wt%.

7. The medicament according to claim 6, characterized in that, The medicament is in the form of a solid dosage form or a solution dosage form, the solid dosage form comprises granules, tablets, capsules, pills, dripping pills, and the solution dosage form comprises oral liquid preparations, intragastric administration preparations, injection administration dosage forms.

8. The medicament according to claim 7, characterized in that, The solution dosage form is a solution of water and the retinene, or a solution of normal saline and the retinene.

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