阅读说明：本技术 柚皮素在制备促进m1型小胶质细胞向m2极化的促进剂中的应用 (Application of naringenin in preparing promoter for promoting M1 microglia to polarize to M2 ) 是由 杨志友 张永平 宋采 邓嘉航 丰心月 马智慧 于 2021-10-13 设计创作，主要内容包括：本发明公开了柚皮素在制备促进M1型小胶质细胞向M2极化的促进剂中的应用。本发明研究表明,柚皮素促进Aβ诱导的M1型小胶质细胞向M2极化,柚皮素的加入激活了M2型小胶质细胞的数量,从而高表达了Aβ降解酶,进一步研究表明,柚皮素是通过上调PPARγ的表达参与了其诱导M2小胶质细胞亚型的转化。柚皮素具有促进小胶质细胞从M1表型向M2表型转化的作用,在抑制炎症反应、清除毒性物质及修复损伤神经等方面具有较大的应用前景。(The invention discloses an application of naringenin in preparing an accelerant for promoting M1 microglia to polarize to M2. The research of the invention shows that naringenin promotes the polarization of the A beta-induced M1 microglia to M2, the addition of naringenin activates the number of the M2 microglia, thereby highly expressing A beta degrading enzyme, and the further research shows that naringenin participates in the transformation of inducing M2 microglia subtype by up-regulating the expression of PPAR gamma. Naringenin has the function of promoting microglia to transform from M1 phenotype to M2 phenotype, and has a wide application prospect in the aspects of inhibiting inflammatory reaction, removing toxic substances, repairing damaged nerves and the like.)

1. Application of naringenin in preparing promoter for promoting M1 microglia to polarize to M2 is provided.

2. Application of naringenin in preparing promoter for promoting expression of A beta degrading enzyme is provided.

3. Application of naringenin in preparing promoter for promoting expression of Abeta degrading enzyme gene is provided.

4. Application of naringenin in preparing medicine for improving memory function of Alzheimer disease patient is provided.

5. The use of claim 1, wherein the M1-type microglia are a β -induced M1-type microglia.

6. The use according to any one of claims 1 to 5, wherein naringenin is used at a concentration of 1 to 100 μ M.

7. The use according to any one of claims 1 to 5, wherein naringenin is used at a concentration of 50 to 100 μ M.

8. An agent for promoting polarization of M1-type microglia to M2, comprising naringenin.

9. The enhancer of claim 8, further comprising a pharmaceutically acceptable excipient.

Technical Field

The invention relates to the technical field of biological medicines, and in particular relates to application of naringenin in preparing an accelerant for promoting M1 microglia to polarize to M2.

Background

Microglia (microglia) is the only cell in nervous tissue derived from mesoderm. There are two types of activation known for microglia. There are pro-inflammatory M1 phenotype microglia and anti-inflammatory M2 phenotype microglia. Proinflammatory M1 phenotype microglia produces interleukin-1 beta (IL-1 beta), tumor necrosis factor (TNF-alpha), Inducible Nitric Oxide Synthase (iNOS), and the like. IL-1 beta is an effector, and microglia cause intracerebral inflammation in the presence of IL-1 beta, and release free radicals such as free radical active oxygen. In addition, in IL-1. beta. producing microglia, the ability to phagocytose metabolic wastes such as aged (damaged) cells is reduced. Thus, the pro-inflammatory M1 phenotype microglia produces damage to nerve cells. And anti-inflammatory M2 phenotype microglia produces arginase-1 (ARG1), interleukin-10 (IL-10), interleukin-4 (IL-4), Transforming Growth Factor (TGF) -beta 1, and the like. ARG1 is an effector that acts to protect nerve cells by releasing ARG 1. The anti-inflammatory M2 phenotype microglia has high capability of phagocytizing metabolic wastes such as aging cells and the like while inhibiting inflammation in brain, and therefore has anti-inflammatory effect, namely protective effect on nerve cells.

US patent US15442473 discloses that the transcription factors Nurr1 and Foxa2 interact with each other to switch glial cells from the M1 phenotype to the M2 phenotype that establishes a therapeutic environment; chinese patent CN112007142A discloses a polar transformation promoter to anti-inflammatory M2 phenotype microglia, which is a novel cyclic peptide derivative. Naringenin is a flavonoid component extracted from dried fruits of Citrus paradisi Macfadyen and Citrus reticulata and Citrus aurantium of Rutaceae, and has antibacterial, antiinflammatory, anticancer, spasmolytic and choleretic effects. Recent experiments show that naringenin has a certain protective effect on cerebral ischemia reperfusion injury and A beta-induced neuron injury; however, there is no report on the role of naringenin in the subtype differentiation of microglia.

Disclosure of Invention

The invention aims to overcome the defects and shortcomings in the prior art and provides application of naringenin in preparing a promoter for promoting polarization of M1 microglia to M2.

The above object of the present invention is achieved by the following technical solutions:

according to the invention, primary microglia are cultured, A beta is added to induce the primary cultured microglia, and then naringenin is added to research the influence of naringenin on the microglia subtype differentiation, and experimental results show that naringenin promotes the polarization of the A beta-induced M1 microglia to M2, the addition of naringenin activates the number of M2 microglia, so that A beta degrading enzyme is highly expressed, and further research shows that naringenin participates in the induction of the M2 microglia subtype transformation by up-regulating the expression of PPAR gamma. The invention therefore claims the following uses for naringenin:

application of naringenin in preparing promoter for promoting M1 microglia to polarize to M2 is provided. Namely the application of naringenin in preparing a promoter for promoting the polarization of proinflammatory M1 phenotype microglia to anti-inflammatory M2 phenotype microglia.

Application of naringenin in preparing promoter for promoting expression of A beta degrading enzyme is provided.

Application of naringenin in preparing promoter for promoting expression of Abeta degrading enzyme gene is provided.

Application of naringenin in preparing medicine for improving memory function of Alzheimer disease patient is provided.

Specifically, the M1 type microglia is an A beta induced M1 type microglia.

Preferably, the naringenin is used at a concentration of 1-100 μ M.

Preferably, the naringenin is used at a concentration of 50-100 μ M.

The invention also provides an accelerant for promoting M1 microglia to polarize to M2, wherein the accelerant comprises naringenin.

Preferably, the enhancer further comprises a pharmaceutically acceptable adjuvant.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides a new application of naringenin in preparing a promoter for promoting M1 microglia to polarize to M2. The research of the invention shows that naringenin promotes the polarization of the A beta-induced M1 microglia to M2, the addition of naringenin activates the number of the M2 microglia, thereby highly expressing A beta degrading enzyme, and the further research shows that naringenin participates in the transformation of inducing M2 microglia subtype by up-regulating the expression of PPAR gamma. Naringenin has the function of promoting microglia to transform from M1 phenotype to M2 phenotype, and has a wide application prospect in the aspects of inhibiting inflammatory reaction, removing toxic substances, repairing damaged nerves and the like.

Drawings

FIG. 1 is the docking site of the Autodock molecules for naringenin and PPAR γ.

Fig. 2 shows that naringenin promotes a β -induced polarization of M1-type microglia to M2. (A) An experimental flow chart; (B) representative immunofluorescent stained pictures; (C) naringin promotes A beta induced CD206^-iNOS⁺CD 206-mediated transformation of M1-type microglia⁺iNOS^-Polarization of M2 type.

Fig. 3 shows that naringenin activates microglia and promotes phagocytosis and degradation of a β. (A) An experimental flow chart; (B) phagocytosis of a β by M1 type microglia; (C) phagocytosis of a β by non-M1 type microglia (D) phagocytosis of a β by M2 type microglia; (E) phagocytosis of a β by non-M2 type microglia.

FIG. 4 shows naringenin up-regulates PPAR γ and IDE, NEP expression in microglia. (A) An experimental flow chart; (B) representative immunofluorescent staining pictures; (C) naringenin up-regulates the expression of PPAR γ in microglia; (D) naringenin up-regulates IDE expression in microglia; (E) naringenin upregulates NEP expression in microglia.

FIG. 5 shows that naringenin up-regulates the expression of IDE, NEP in microglia cells via PPAR γ. (A) An experimental flow chart; (B) naringenin ameliorates a β 1-42 induced reduction in PPAR γ receptor levels; (C) naringenin regulates expression of IDE through PPAR gamma receptor; (D) naringenin regulates the expression of NEP through PPAR gamma receptor.

FIG. 6 shows that naringenin improves cognitive memory function in 5xFAD mice. (A) A flow chart of a behavioristics experiment; (B) weight change in mice during dosing; (C) spontaneous movement of the mouse within 10 min; (D) testing the cognitive memory ability of the object; (E) the object position memorizes energy.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

The chemical structural formula of naringenin is shown as follows:

example 1 molecular docking study of naringenin and its target of action PPAR γ

The method comprises the following steps: the 3D structure of the compound naringenin is obtained from PubChem (https:// PubChem. ncbi. nlm. nih. gov /), the crystal structure of the core target protein is obtained from RCSB PDB (http:// www.rcsb.org /) database by screening, and the structure of the protein is separated by removing water molecules and ligand small molecules in the protein by pymol (version 2.4.0). The method is characterized in that a genetic algorithm in AutoDock 4.2.6 software is adopted for semi-flexible docking, the central coordinates and the size of a box are set according to the position of an active site of a protein molecule and a region where the protein molecule possibly acts on a ligand small molecule, other parameters are kept default, and molecular docking analysis is carried out on a compound and a target protein.

As a result: the molecular docking is to simulate the interaction between the ligand micromolecules and the receptor biomacromolecules, obtain the optimal binding position and binding strength of the ligand micromolecules and predict the affinity of the ligand micromolecules and the receptor biomacromolecules. Molecular docking analysis is carried out on naringenin and potential target PPAR gamma thereof, conformation with lower binding energy among molecules is more stable, the molecular conformation with low binding energy in the screening docking result is the optimal docking result, and the binding sites of the optimal docking result are analyzed. The results of docking are shown in FIG. 1, which shows that naringenin binds to amino acid residues Glu259 in the PPAR γ receptor macromolecule,Arg280, Arg288 and Ser342 can have good binding force, and the binding energy is-7.21 kcal & mol^-1Indicating that it has good affinity.

Example 2 naringenin promotes polarization of M1 microglia to M2

Primary microglia cell culture: 5-6 ddY mice (P2-4), cerebral cortex isolation after alcohol sterilization, dura mater removal, chopping in DMEM (+ FBS) medium, centrifugation (1000rpm,3min), supernatant removal, addition of 2mL of 0.25% trypsin at 37 degrees, 5% CO₂The cells were incubated for 30min, 4mL of 10% FBS-DMEM medium was added, centrifuged (2000rpm,5min), the supernatant removed, 2mL of a DNase I/trypsin inhibitor was added, and 10% CO was added at 37 degrees₂The culture chamber of (1) was incubated for 15min, 4mL of 10% FBS-DMEM medium was added, centrifugation (1000rpm,3min) was performed, the supernatant was removed, 4mL of 10% FBS-DMEM medium was added, the dissociated cells were blown with a rubber-tipped pipette, 2mL of 15% BSA in PBS was added from the bottom, centrifugation (2000rpm,6min) was performed, the supernatant was removed, 2mL of 10% FBS-DMEM medium was added, and the cells were counted and plated on 6-cm culture dishes (8 × 10)⁵Individual cells). After 14 days of culture, when the cells reached confluency, microglia were separated, rinsed once with PBS, and serum-free DMEM 3mL and 0.25% trypsin 1mL were added at 37 degrees, 5% CO₂The incubator of (1) was incubated for 1h, the supernatant was removed, rinsed once with PBS, and 2mL of 0.25% Trypsin was added at 37 ℃ in 5% CO₂The cells were incubated in the incubator for 5min, 4mL of 10% FBS-DMEM was added, the dissociated cells were blown out with a rubber-tipped dropper, and the cells were counted and plated on 8-well cell culture plates.

The test method comprises the following steps: experimental procedure primary cultured microglia cells were seeded on 8-well plates (1.5 x 10) as shown in figure 2A⁴cells/well), after 2h, Abeta 1-42 (1. mu.M) was added for 24h, after which the supernatant containing Abeta was removed, a blank control (DMSO), a positive control (IL-4) and naringenin were added, after 24h incubation, the cells were fixed with 4% PFA, immunofluorescent staining was performed with the addition of the primary antibodies CD206(1:200, M2 marker) and iNOS (1:50, M1 marker) and the secondary antibodies Alexa Fluor 488 and 594(Invitrogen, Carlsbad, CA, USA), photographs were taken with a fluorescence microscope (BX-61/DP70, Olympus) and image analysis was performed with Metamorph software.

The test results are shown in fig. 2B-C, which show that naringenin (50-100 μ M) can significantly reduce M1 and increase the proportion of M2 microglia, and naringenin tenders the polarization of a β -induced CD206+ iNOS-M1 microglia to CD206-iNOS + M2, indicating that naringenin promotes a β -induced M1 microglia to M2.

Example 3 naringenin activates microglia phagocytosis of A β

The method comprises the following steps: experimental procedure Primary cultured microglia cells were seeded in 8-well plates (1.5 x 10) as shown in FIG. 3A⁴cells/well), 1h later, adding naringenin (0.1-100 μ M) or positive control IL4, culturing for 4h later, adding Abeta 1-42(1 μ M) for further culturing for 24h, removing supernatant containing Abeta and naringenin, adding Abeta 1-42Hilyte Fluor 488, culturing for 3h later, fixing cells with 4% PFA, adding primary antibody CD206(1:200, M2 marker) or iNOS (1:50, M1 marker) and secondary antibody Alexa Fluor 594(Invitrogen, Carlsbad, CA, USA) for immunofluorescence staining, taking pictures with a fluorescence microscope (BX-61/DP70, Olympus) and analyzing images with Metamorph software.

As shown in fig. 3B, after the addition of a β 1-42, the phagocytic capacity of the fluorescent-labeled a β 1-42Hilyte Fluor 488 by the microglia is greatly increased, but with the increase of naringenin concentration, the number of CD 206-positive microglia containing the fluorescent-labeled a β 1-42 is significantly reduced while the number of iNOS-positive microglia is unchanged, and the result may suggest that the addition of naringenin activates the number of M2-type microglia, thereby highly expressing a β degrading enzyme to degrade exogenous foreign substances, while the degradation of M1-type microglia is weak.

Example 4 Regulation of A β -degrading enzyme expression in microglia by Naringin

The method comprises the following steps: the experiment firstly discusses the influence of naringenin on PPAR gamma expression, the experimental flow is shown in figure 4A, BV2 microglia is inoculated on an 8-well plate (3000cells/well), naringenin (1-100 muM) or positive control pioglitazone (PPAR gamma agonist) is added after 24h, after further culturing for 24h, the cells are fixed by 4% PFA, primary antibodies IDE (1:500) or NEP (1:100) and PPAR gamma (1:100) and secondary antibodies Alexa Fluor 594 and 488(Invitrogen, Carlsbad, CA, USA) are added for immunofluorescence staining, a fluorescence microscope (BX-61/DP70, Olympus) is used for photographing, and image analysis is carried out by Metamorph software.

The results are shown in FIGS. 4B-E: the up-regulation of naringenin concentration dependency on the expression of PPAR gamma, A beta degrading enzymes IDE and NEP in BV2 microglia, and the expression of IDE and NEP in cells with high PPAR gamma expression are also obviously increased, which shows that the up-regulation of the PPAR gamma expression by naringenin is likely to participate in the transformation of inducing M2 microglia subtype.

Example 5 PPAR γ inhibitors abrogating the Up-regulating Effect of naringenin on A β -degrading enzymes in microglia

The method comprises the following steps: experimental protocol as shown in figure 5A BV2 microglia were seeded on 8-well plates (3000cells/well) with GW9662(2 μ M) added after 24h, naringenin (10 and 50 μ M) or positive control pioglitazone (PPAR γ agonist 10 μ M) added after 0.5h, Α β 1-42(1 μ M) added after 1h, cells were fixed with 4% PFA after further incubation for 24h, immunofluorescent staining was performed with primary antibodies IDE (1:500) or NEP (1:100) and PPAR γ (1:100) and secondary antibodies Alexa fluors 594 and 488(Invitrogen, Carlsbad, CA, USA), photographs were taken with a fluorescence microscope (BX-61/DP70, Olympus) and image analysis was performed with Metamorph software.

The results showed that naringenin ameliorated a β 1-42 induced reduction in PPAR γ receptor levels (fig. 5B); GW9662 was found to abolish naringenin-induced increases in IDE and NEP expression using the PPAR γ inhibitor GW9662 (fig. 5C-D), indicating that naringenin participates in its induction of M2 microglial subtype transformation by up-regulating PPAR γ expression.

Example 6 Naringin improves 5XFAD mouse memory function

Naringenin (5,100mg/kg/day) was orally administered to AD model mice 5XFAD (8-12 months old), spontaneous locomotor test was performed 21 days later, object cognitive memory test (ORT) was performed 22 days later, and object position memory test (OLT) was performed 29 days later.

Spontaneous activity test: the mice are placed in a spontaneous activity device (the length is 33cm, the width is 28cm, and the height is 26.5cm), the activity process within 10min is shot by a camera and input into a computer, and the total route of the mice is automatically recorded and analyzed by an animal behavior analysis system.

Cognitive memory testing of new objects: during training, two identical objects A and A ' were placed in an autonomous mobile device (33 cm long, 28cm wide, 26.5cm high), and then a mouse was placed in the device, and the mouse was allowed to spontaneously explore the objects A and A ', and the number of times of exploration (sniffing the nose and climbing the paw) was recorded, and the probability of exploring the object A ' by the mouse was calculated as a percentage of the total number of times of exploration A ' in A, A ', i.e., A '/(A + A ') × 100%. After 48h, the object A' in the movable box was taken out, changed to a different novelty object B, and the mouse was placed in it again, allowing the mouse to explore the objects A and B spontaneously, and recording the number of times of exploration. The probability B/(a + B) × 100% that the mouse explores object B is calculated, see fig. 6C, the test being based on the preference of the mouse to contact the novelty object.

And (3) position memory test: during training, different patterns are attached to two sides of the activity box, two identical objects A1 and A2 are placed in the spontaneous activity box, then a mouse is placed in the spontaneous activity box, the mouse spontaneously explores the objects A1 and A2, the exploration times are recorded, and the probability of exploring the object A1 by the mouse is calculated. After 48h, the object a1 in the movable box was changed to another position a1 'in the movable box, and the mouse was again placed in it, and allowed to spontaneously explore the objects a 1' and a2, and the number of times of exploration was recorded. The probability of the mouse exploring a1 ' a1 '/(a 1 ' + B) × 100% was calculated, see fig. 6A, the test being based on the mouse preferring to touch objects at the new location.

The statistical method comprises the following steps: experimental data are expressed as mean ± SEM, and analysis of variance of the two-way Bonferroni test between groups was performed using Graphpad prism5.0 software.

The experimental results are shown in fig. 6B-E, which show that the spontaneous exercise of mice is not affected after the administration of naringenin, and the high concentration of naringenin can significantly improve the cognitive memory function of 5XFAD mice and has the tendency of increasing position memory.