Application of 1-O-acetyl-4R, 6S-inula flower lactone in preparation of medicine for preventing and treating neuroinflammation

Abstract

The invention discloses an application of 1-O-acetyl-4R, 6S-inula flower lactone in preparation of a medicine for preventing and treating neuroinflammation, and researches find that 1-O-acetyl-4R, 6S-inula flower lactone (AB) is connected with kinase by targeting PDZ, so that inflammatory response is reduced, oxidative stress is reduced, mitochondrial balance can be maintained, and neuroinflammation is inhibited; in a cell experiment, AB can inhibit lipopolysaccharide induced BV-2 microglial cell activation, reduce release of inflammatory factors such as microglial cell tumor necrosis factor-alpha and interleukin-6, inhibit mitogen activated protein kinase and influence an AMP dependent protein kinase signal channel; in animal experiments, AB can relieve mouse neuroinflammation induced by LPS and improve brain neuron damage. AB can be used for developing medicines for preventing and treating neuroinflammation, has no obvious toxic and side effects, and has the advantages of low dosage, safety, oral administration and the like.

Description

Technical Field

[0001] The invention relates to the field of medical technology, and in particular to application of 1-O-acetyl-4R, 6S-inula lactone in the preparation of drugs for preventing and treating neuroinflammation. Background Art

[0002] Neuroinflammation is a type of disease caused by abnormal activation of immune cells in the central nervous system (CNS), with clinical manifestations including limb numbness, autonomic dysfunction, and impaired consciousness. Long-term neuroinflammation can lead to a series of complications, such as paresthesia, cognitive impairment, and other neurological diseases (such as Parkinson's disease), leading to limb paralysis, epilepsy, and multiple organ dysfunction.

[0003] Neuroinflammation can be caused by a variety of factors, including mechanical injury, infection, metabolic disorders, and poisoning. Currently, commonly used clinical treatment drugs include the immunomodulatory drug dexamethasone, the nutritional support drug citicoline, and the analgesics ibuprofen and carbamazepine. However, existing treatments have limited effects, large side effects, and usually can only relieve symptoms, making it difficult to achieve complete cure or long-term control of the disease.

[0004] Therefore, finding new therapeutic targets for neuroinflammation is of great significance for the development of drugs for the prevention and treatment of neuroinflammation.

[0005] 1-O-acetyl-4R, 6S-britannilactone (AB) is a compound isolated from Inula japonica. So far, only one study has reported the pharmacological activity of AB in inhibiting LPS-mediated NO production. Currently, the activity research of AB is relatively limited, and there are no reports on the application of AB in the treatment of neuroinflammation and the effective targets of AB. Summary of the invention

[0006] The purpose of the present invention is to overcome the deficiencies of the prior art and provide an application of 1-O-acetyl-4R, 6S-inula lactone in the preparation of drugs for preventing and treating neuroinflammation.

[0007] The second object of the present invention is to provide a use of a pharmaceutical preparation containing 1-O-acetyl-4R, 6S-inula lactone in the preparation of drugs for preventing and treating neuroinflammation.

[0008] The technical solution of the present invention is summarized as follows:

[0009] Application of 1-O-acetyl-4R,6S-inula lactone in the preparation of drugs for preventing and treating neuroinflammation.

[0010] Application of a pharmaceutical preparation containing 1-O-acetyl-4R,6S-inula lactone in the preparation of drugs for preventing and treating neuroinflammation.

[0011] Advantages of the present invention:

[0012] The present invention found that 1-O-acetyl-4R, 6S-britannilactone (AB) reduces inflammatory response, reduces oxidative stress, and maintains mitochondrial balance by targeting PDZ-linked kinase (PBK, Gene ID: 52033), thereby inhibiting neuroinflammation.

[0013] In cell experiments, AB was able to inhibit lipopolysaccharide (LPS)-induced BV-2 microglia activation, reduce the release of inflammatory factors such as microglial tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), and inhibit mitogen-activated protein kinase (MAPK) and affect AMP-dependent protein kinase (AMPK) signaling pathways.

[0014] In animal experiments, AB can alleviate LPS-induced neuroinflammation in mice and improve brain neuron damage. AB can be used to develop drugs for the prevention and treatment of neuroinflammation, and no obvious toxic side effects have been found. AB has the advantages of low dose, safety, and oral administration. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 AB showed anti-neuroinflammatory effects in vitro by affecting MAPK and AMPK pathways;

[0016] in:

[0017] A is the structure of 1-O-acetyl-4R,6S-inula lactone (AB);

[0018] B AB inhibits the production of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in BV-2 cells induced by LPS

[0019] Students (n=3);

[0020] C AB inhibited the mRNA levels of inflammatory factors IL-6, IL-1α, IL-1β, CCL5, CXCL10, MCP-1, and TNF-α in BV-2 cells induced by LPS (n=3);

[0021] D AB inhibited the expression of microglial activation marker Iba-1 and macrophage surface marker CD68 in BV-2 cells induced by LPS;

[0022] E Flow cytometry verified that AB restored the changes in mitochondrial membrane potential of BV-2 cells induced by LPS;

[0023] F Flow cytometry verified that AB inhibited the expression of IL-6 in BV-2 cells induced by LPS;

[0024] Effects of G AB on the protein levels of MAPK and AMPK pathways in LPS-induced BV-2 cells (n=3).

[0025] Figure 2 AB can alleviate LPS-mediated neuroinflammation in vivo, including:

[0026] (A) Effect of AB on the movement trajectory of mice in the Morris water maze test;

[0027] (B) AB enhanced the movement speed and distance of LPS-stimulated mice in the Morris water maze test (n=8);

[0028] (C) AB inhibited p62 immunofluorescence staining in brain tissue of LPS-stimulated mice;

[0029] (D) IHC staining of microglial marker Iba-1 and macrophage surface marker CD68 in brain tissue of mice with neuroinflammation;

[0030] (E) Effects of AB on the mRNA levels of IL-6 and IL-1α in the brain tissue of LPS-stimulated mice (n=4);

[0031] (F) Effects of AB on the levels of MAPK pathway proteins in brain tissue of LPS-stimulated mice (n=3);

[0032] (G) Effects of AB on the protein levels of AMPK pathway in brain tissue of LPS-stimulated mice (n=3).

[0033] Figure 3 PBK is a potential target of AB, among which:

[0034] (A) Biotin-coupled AB (Bio-AB) was used to probe the Huprot proteome microarray;

[0035] (B) Microscale thermophoresis MST detection of AB and PBK binding affinity;

[0036] (C) Biotin pull-down assay, and bound proteins were detected by immunoblotting (n = 3);

[0037] (D) Drug affinity response target stability experiment of AB and PBK (n=3);

[0038] (E) Protein thermal stability analysis experiment of AB and PBK (n=3).

[0039] Figure 4 Knockout of PBK impaired the anti-neuroinflammatory effects of AB in vitro, including:

[0040] (A) PBK knockout abolished the effect of AB on the mRNA levels of IL-6, IL-1α, IL-1β, and iNOS in LPS-induced BV-2 cells (n=3);

[0041] (B) Flow cytometry results showed that PBK knockdown abolished the effect of AB on IL-6 levels;

[0042] (C) COX-2 and p62 immunofluorescence staining;

[0043] (D) PBK knockout abolished the effects of AB on MAPK and AMPK signaling pathways and mitochondrial fusion proteins (n=3).

[0044] Figure 5 This is the hydrogen spectrum of 1-O-acetyl-4R,6S-inula lactone.

[0045] Figure 6 This is the carbon spectrum of 1-O-acetyl-4R,6S-inula lactone. DETAILED DESCRIPTION

[0046] The technical scheme of the present invention is described in detail below in conjunction with specific embodiments. The embodiments are only examples of a part of the present invention, not all embodiments. Based on the embodiments of the present invention, any other embodiments made by any person skilled in the art without creative work should belong to the protection scope of the present invention.

[0047] For the parts where specific experimental steps or conditions are not clearly stated in the examples, standard experimental procedures or conditions described in conventional literature in the art can be referred to. If the manufacturer of the reagents or instruments used is not indicated, they are all commercially available conventional reagent products, and the corresponding suppliers can be selected as needed.

[0048] Example 1

[0049] At the cellular level: AB blocked LPS-induced BV-2 microglial activation, inhibited inflammatory response and alleviated mitochondrial damage in vitro by inhibiting MAPK signaling pathway activation.

[0050] (1) Experimental Materials

[0051] Mouse TNF-α and IL-6 levels were measured using the corresponding enzyme-linked immunosorbent assay (ELISA) kits (Xinbosheng Biotechnology Co., Ltd.);

[0052] Mouse microglia (BV-2, Wuhan Pronocell Life Science Co., Ltd.).

[0053] The 1-O-acetyl-4R, 6S-britannilactone (AB) used in this experiment was extracted from Inula britannica L. by successively extracting it with 95% ethanol aqueous solution and 75% ethanol aqueous solution for 2 hours, twice, and then passing the ethyl acetate extract through a silica gel column, eluting with dichloromethane and methanol (100:1-1:1) to obtain AB and other fractions, and preparing liquid phase purification using 1 H and 13 Its structure was confirmed by C NMR. Figure 5 The hydrogen spectrum of 1-O-acetyl-4R,6S-inula lactone ( 1 H(600MHz,CDCl 3 )NMR spectrum of AB.); Figure 6 The carbon spectrum of 1-O-acetyl-4R,6S-inula lactone ( 13 C(150MHz,CDCl 3 )NMR spectrum of AB). The structure identified is shown in Figure 1 A.

[0054] Bio-AB is biotin-coupled 1-O-acetyl-4R, 6S-britannilactone (AB).

[0055] The primary antibodies used in immunoblotting experiments included p-ERK (Proteintech, 28733-1-AP, 1:1000), ERK (Abmart, T40071F, 1:1000), p-JNK (Abcolonl, AP1337, 1:1000), JNK (Proteintech, 66210-1-Ig, 1:1000), p-p38 (Abmart, TP56391F, 1:1000), p38 (Proteintech, 14064-1-AP, 1:1000). 1000),p-AMPK(Abcolonl,AP1002,1:1000),AMPK(Proteintech,10929-2-AP,1:2000),p-mTOR(Proteintech,67778-1- Ig1:2000,mTOR(Proteintech,66888-1-Ig,1:5000),p62(Abcolonl,A19700,1:20000),iNOS(Proteintech,22226-1-AP 1:1000), Mfn1 (Proteintech, 13798-1-AP, 1:1000), Drp1 (Proteintech, 12957-1-AP, 1:2000), PBK (Abcam, ab236872 1:1000), and GAPDH (Proteintech, 60004-1-Ig 1:4000), the primary antibodies were diluted in TBST containing 5% BSA.

[0056] The secondary antibodies used in immunoblotting include goat anti-rabbit recombinant secondary antibody (Proteintech, RGAR001, 1:4000) and goat anti-mouse recombinant secondary antibody (Proteintech, RGAM001, 1:4000).

[0057] (2) Experimental methods

[0058] 1) BV-2 cells were cultured in a medium (DMEM) containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% CO2. Before stimulation with lipopolysaccharide (LPS, 500 ng / mL), cells were pretreated with different concentrations of AB according to experimental needs. After 24 hours of stimulation, the cells were centrifuged and the BV-2 cell supernatant and BV-2 cell pellet were collected for subsequent detection;

[0059] 2) ELISA:

[0060] According to the instructions of mouse TNF-α ELISA kit (Xinbosheng Biotechnology Co., Ltd.) and mouse IL-6 ELISA kit (Xinbosheng Biotechnology Co., Ltd.), the BV-2 cell supernatant obtained in experimental method 1) was taken to detect the content of TNF-α and IL-6 in the supernatant; see Figure 1 B, The results showed that AB significantly inhibited the secretion of LPS-induced proinflammatory cytokines IL-6 and TNF-α, showing an anti-inflammatory effect.

[0061] 3) Real-time quantitative fluorescence PCR:

[0062] RNA was extracted from BV-2 cells using TRIzol reagent and The quantity and purity were assessed using an N50 spectrophotometer. Reverse transcription was performed using a reverse transcription system. The levels of target mRNA were quantified by real-time fluorescence PCR using Applied Biosystems QuantStudio1, with GAPDH as an internal reference gene; see Figure 1 C, AB downregulated the mRNA levels of IL-6, IL-1α, IL-1β, CCL5, CXCL1, MCP-1, and TNF-α in a dose-dependent manner.

[0063] 4) Immunofluorescence:

[0064] BV-2 cells were inoculated into a twelve-well plate, and AB was added at a final concentration of 20 μM. After AB acted for 1 hour, LPS was added at a final concentration of 500 ng / mL. After 4 hours, BV-2 cells were subjected to immunofluorescence staining. BV-2 cells were fixed with a 4% paraformaldehyde solution (solvent was PBS) at room temperature for 10 minutes, washed 3 times with phosphate buffer (PBS), permeabilized with 0.1% TritonX-100 for 10 minutes, blocked with 5% BSA at room temperature for 90 minutes, and then incubated overnight at 4°C with the corresponding primary antibody. Washed 3 times with PBS, incubated at room temperature with a secondary antibody labeled with a fluorescent dye of the corresponding properties and DAPI staining, and sealed with an anti-fluorescence quenching sealing agent. The sections were observed by a LeicaDM6B microscope (Leica, Germany); see Figure 1 D, Experimental results showed that AB reduced the expression of inflammation-related markers Iba-1 and CD68.

[0065] 5) Flow cytometry:

[0066] In the mitochondrial membrane potential flow cytometry analysis, BV-2 cells were pretreated with 20 μM AB for 1 hour and stimulated with LPS for 24 hours, and then the BV-2 cell pellets were collected. According to the instructions of Fluo-4, AM calcium fluorescent probe Fluo-4, AM (Solabo Biotechnology Co., Ltd.), the staining was performed, and the cells were filtered and then passed through a CytoFlex flow cytometer (Beckman, USA). The positive cell population was analyzed; given the effect of inflammation on mitochondrial homeostasis, we observed that AB could significantly improve mitochondrial membrane potential, see Figure 1 E.

[0067] BV-2 cells were seeded into six-well plates. BV-2 cells were pretreated with 20 μM AB for 1 hour and stimulated with LPS for 24 hours. BV-2 cell pellets were collected. The cells were washed with PBS, fixed with 4% paraformaldehyde solution, permeabilized with 0.1% Triton X-100, blocked with 5% BSA at 4°C for 40 minutes, incubated with primary antibody IL-6 for 2 hours, washed with PBS, incubated with fluorescent dye-labeled R secondary antibody for 1 hour, washed with PBS, filtered, and then analyzed by CytoFlex flow cytometer (Beckman, USA); see Figure 1 F, Flow cytometric analysis further verified the inhibitory effect of AB on IL-6 production.

[0068] 6) Western Blot:

[0069] Western and IP cell lysis buffer (Biyuntian Biotechnology Co., Ltd.) was used to lyse BV-2 cell pellets to extract total cell protein, and the protein concentration was determined using a BCA protein concentration assay kit (Biyuntian Biotechnology Co., Ltd.). Equal masses of protein were separated by 10% SDS-PAGE gel electrophoresis and transferred to a PVDF membrane, blocked with 5% skim milk at room temperature for 2 hours, and the antibody was diluted with 5% BSA as a diluent, and incubated with the primary antibody at 4°C overnight. After washing three times with Tris-HCl buffered saline (TBST) containing 1‰ Tween-20, the corresponding secondary antibody was incubated at room temperature for 2 hours, washed three times with TBST, and developed with enhanced chemiluminescence solution (ECL); Figure 1 As shown in G, AB can effectively reverse the activation of MAPK and AMPK signaling pathways induced by LPS stimulation and inhibit the expression of inflammatory factor iNOS.

[0070] 7) Statistical analysis:

[0071] The data were analyzed using GraphPad Prism 8 (GraphPad Software Inc, San Diego, CA, USA). The data are shown as mean ± standard error of the mean, and one-way analysis of variance was used for statistical analysis. P < 0.05 was considered statistically significant.

[0072] (3) Experimental results

[0073] The above results indicate that 1-O-acetyl-4R, 6S-britannilactone (AB) exerts its anti-neuroinflammatory effect by inhibiting LPS-induced MAPK and AMPK pathway activation in vitro.

[0074] Example 2

[0075] Animal level: AB alleviates LPS-induced neuroinflammation in mice.

[0076] (1) Experimental Materials

[0077] Specific pathogen-free male C57BL / 6 mice (23-25 ​​g, 8 weeks old) were purchased from the market. The room temperature was 22-24°C, the relative humidity was 50%-65%, the ventilation was good, the artificial day and night (12h / 12h), and the food and water were freely available.

[0078] Antibodies used for immunoblotting included p-ERK (1:1000), ERK (1:1000), p-JNK (1:1000), JNK (1:10000), p-p38 (1:1000), p38 (1:1000), p-AMPK (1:1000), AMPK (1:1000), p-mTOR (1:2000), mTOR (1:5000), p62 (1:20000), and GAPDH (1:4000).

[0079] (2) Experimental methods

[0080] 1) Modeling of Neuroinflammation Mice

[0081] Male C57BL / 6 mice were randomly divided into:

[0082] Blank control group, blank drug administration group, LPS model group, low-dose treatment group, and high-dose treatment group.

[0083] The blank control group was gavaged with the same volume of 10% hydroxypropyl-β-cyclodextrin aqueous solution as the blank administration group for 7 consecutive days, and the same volume of injection saline as the model group was intraperitoneally injected on the third and fourth days.

[0084] The blank group was intragastrically administered with AB (50 mg / kg / day, dissolved in 10% hydroxypropyl-β-cyclodextrin aqueous solution) for 7 consecutive days, and the same volume of saline as that of the model group was intraperitoneally injected on the third and fourth days.

[0085] The LPS model group was intragastrically administered with the same volume of 10% hydroxypropyl-β-cyclodextrin aqueous solution as the blank administration group for 7 consecutive days, and LPS (concentration of 5 mg / kg, dissolved in physiological saline) was intraperitoneally injected on the third and fourth days.

[0086] The low-dose treatment group was given AB (10 mg / kg / day, dissolved in 10% hydroxypropyl-β-cyclodextrin aqueous solution) by oral gavage for 7 consecutive days. LPS (concentration of 5 mg / kg, dissolved in normal saline) was injected intraperitoneally on the third and fourth days.

[0087] The high-dose treatment group was given AB (50 mg / kg / day, dissolved in 10% hydroxypropyl-β-cyclodextrin aqueous solution) by oral gavage for 7 consecutive days. LPS (concentration of 5 mg / kg, dissolved in normal saline) was injected intraperitoneally on the third and fourth days.

[0088] Morris water maze test on the seventh day

[0089] After the experiment, the mice were killed and the brain tissues were taken for testing.

[0090] 2) Morris water maze

[0091] The experimental apparatus includes a circular water maze pool (inner diameter 120 cm, height 50 cm), a video tracking system, and related data analysis software. The water temperature was maintained at 23±2℃. The video tracking software divided the pool into four quadrants, and the platform was located in the second quadrant. The mice were placed in the pool, facing the walls of the four quadrants, and given 90 seconds to find the platform. If they could not find the platform during this period, they were gently guided to the platform for 30 seconds and then removed from the platform. In the spatial exploration test, the platform was removed from the pool, and the mice were gently placed in the four quadrants respectively, allowed to swim freely in the pool for 90 seconds to find the platform, and the experimental data were recorded. Data analysis was performed using the software provided by Shanghai Yuyan; see. Figure 2 A, B, The classic LPS-induced neuroinflammation mouse model was used. The experimental results showed that mice treated with AB showed significant improvement in motor ability in the Morris water maze test, with a significant increase in both movement speed and movement distance.

[0092] 3) Immunofluorescence staining: After dewaxing and hydration, the paraffin sections were antigen-recovered using a citric acid buffer with a pH of 6.0. Endogenous peroxidase was blocked with an endogenous peroxidase inhibitor for 10 minutes at room temperature in the dark. The sections were blocked with 5% BSA for 90 minutes at room temperature and then incubated with the primary anti-p62 antibody at 4°C overnight. After incubation, the tissues were washed with PBS. Then, the sections were stained with a fluorescent dye-labeled secondary antibody and DAPI at room temperature in the dark. The sections were observed with a Leica DM6B microscope (Leica, Germany); see Figure 2 C, Immunofluorescence results showed that AB treatment significantly inhibited the expression level of p62.

[0093] 4) Immunohistochemical staining: After dewaxing and hydration, the paraffin sections were antigen repaired with a citric acid buffer with a pH of 6.0. Endogenous peroxidase was blocked with an endogenous peroxidase inhibitor (Zhongshan Jinqiao Biotechnology Co., Ltd.) for 10 minutes at room temperature in the dark. Blocked with 5% BSA at room temperature for 90 minutes, incubated with primary antibodies Iba-1 and CD68 at 4°C overnight. Incubated with secondary antibodies (Zhongshan Jinqiao Biotechnology Co., Ltd.) at room temperature for 90 minutes, added with DAB color developer (Zhongshan Jinqiao Biotechnology Co., Ltd.) for color development, stained with hematoxylin for cell nuclei, and rinsed with tap water to turn blue. After dehydration, the sections were sealed with neutral gum. The sections were analyzed with Leica DM6B (Leica, Germany); see Figure 2 D, Immunohistochemical staining showed that AB inhibited the activation of microglia and macrophages.

[0094] 5) Real-time quantitative fluorescence PCR analysis:

[0095] RNA was extracted from brain tissue using TRIzol reagent and The quantity and purity were assessed using an N50 spectrophotometer. Reverse transcription was performed using a reverse transcription system. The levels of target mRNA were quantified by real-time PCR using an Applied Biosystems QuantStudio 1, with GAPDH as an internal reference gene; see Figure 2 E, AB reduced the mRNA levels of inflammatory factors IL-6 and IL-1α.

[0096] 6) Immunoblotting

[0097] 20 mg of brain tissue samples were weighed from each group. The homogenized tissue was lysed using Western and IP cell lysis buffer (Biyuntian Biotechnology Co., Ltd.) to extract the total protein of the tissue. After centrifugation (12000g, 4°C, 10min), the protein concentration of the supernatant was determined using the BCA protein concentration assay kit (Biyuntian Biotechnology Co., Ltd.). Equal amounts of protein were separated by 10% SDS-PAGE gel electrophoresis and transferred to a PVDF membrane. The membrane was blocked with 5% skim milk at room temperature for 2h, and the antibody was diluted with 5% BSA as a diluent. The membrane was incubated with the primary antibody at 4°C overnight. After washing with PBS three times, the secondary antibody of the corresponding resistance was incubated at room temperature for 2h. After washing with PBS three times, the membrane was developed with enhanced chemiluminescence solution. Figure 2 F, G The results showed that AB effectively reversed the activation of MAPK and AMPK signaling pathways.

[0098] 7) Statistical analysis

[0099] The data were analyzed using GraphPad Prism 8 (GraphPad Software Inc, San Diego, CA, USA). The data are shown as mean ± standard error of the mean, and one-way analysis of variance was used for statistical analysis. P < 0.05 was considered statistically significant.

[0100] (3) Experimental results

[0101] These results indicate that AB restored the motor ability of mice and alleviated LPS-mediated neuroinflammation in mice through the MAPK and AMPK pathways.

[0102] Example 3

[0103] At the molecular and cellular level: AB targets PBK, and knocking down PBK eliminates the anti-neuroinflammatory effect of AB in vitro.

[0104] (1) Experimental Materials

[0105] See Example 1, step (1)

[0106] (2) Experimental methods

[0107] 1) BV-2 cells were cultured in a medium (DMEM) containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% CO2. Before stimulation with lipopolysaccharide (LPS, 500 ng / mL), cells were pretreated with different concentrations of AB according to experimental needs. After 24 hours of stimulation, the cells were centrifuged and the BV-2 cell supernatant and BV-2 cell pellet were collected for subsequent detection;

[0108] 2) Proteomic chip target identification:

[0109] Target identification of AB HuProt proteome chip (CDIHP-004) was purchased from Beijing Protein Innovation Company (Beijing, China). HuProt proteome chip was blocked with blocking buffer for 1 h at room temperature. Bio-AB (10 μM) was incubated with the proteome chip at room temperature for 1 h, and then the chip was washed three times with PBS and incubated with Cy5-conjugated streptavidin for 1 h. Figure 3 A, The results show that the protein with the highest signal-to-noise ratio is PBK.

[0110] 3) Micro-thermophoresis:

[0111] The PBK protein was labeled using the Monolith NT kit. Different concentrations of AB were added to the buffer containing the labeled PBK protein (200 nM). After incubation for 15 min, the Kd value of AB and PBK was detected and analyzed using the Monolith NT.115 instrument (Nano-Temper Technologies, München, Germany). Figure 3 B, Microscale thermoelectrophoresis experiments further showed that AB and PBK have a high binding affinity.

[0112] 4) Biotin Pulldown:

[0113] Western and IP cell lysis buffer (Biyuntian Biotechnology Co., Ltd.) was used to lyse BV-2 cell pellets to extract total cell protein, and the protein concentration was determined using the BCA protein concentration assay kit (Biyuntian Biotechnology Co., Ltd.). The cell lysate was incubated with AB at 4°C overnight, incubated with Bio-AB at room temperature for 3 hours the next day, and then incubated with streptavidin magnetic beads (Biyuntian Biotechnology Co., Ltd.) at room temperature for 3 hours. Elution was performed using PBS buffer containing 1% Tween 20. Immunoblotting was then performed; see Figure 3 C, The results of the biotin pulldown experiment showed that the biotin-coupled AB could effectively capture PBK in the cell lysate, while this binding was significantly blocked when an excess of AB was added.

[0114] 5) Drug affinity reaction targeting stability:

[0115] The BV-2 cell precipitate obtained in Example 1 was lysed using Western and IP cell lysis buffer (Biyuntian Biotechnology Co., Ltd.) to extract total cell protein, and the protein concentration was determined using the BCA protein concentration assay kit (Biyuntian Biotechnology Co., Ltd.). The lysate was incubated with (5, 10 and 20 μM AB) for 1 hour at room temperature. Then 1 μg / ml Pronase E (Solebao Biotechnology Co., Ltd.) was added, and the mixture was incubated at 37°C for 5 minutes. The supernatant was collected by centrifugation and subjected to immunoblotting analysis using PBK antibody; see Figure 3 D. Protein thermal stability analysis results show that binding to AB can stabilize PBK

[0116] 6) Cell thermal migration analysis:

[0117] The BV-2 cell precipitate obtained in Example 1 was lysed using Western and IP cell lysis buffer (Biyuntian Biotechnology Co., Ltd.) to extract total cell protein, and the protein concentration was determined using the BCA protein concentration assay kit (Biyuntian Biotechnology Co., Ltd.). The lysate was incubated with 20 μM AB at room temperature for 1 hour, and then heated at different temperatures (42, 46, 50, 54, 58) for 3 minutes. The supernatant obtained after centrifugation was subjected to immunoblot analysis using PBK antibody. Dimethyl sulfoxide (DMSO) was used as the control group of this study. See Figure 3 E, The results of drug affinity reaction targeting stability show that AB can stabilize PBK after binding with PBK and resist the influence of temperature on its stability.

[0118] 7) Real-time quantitative fluorescence PCR analysis:

[0119] RNA was extracted from cells using TRIzol reagent and The quantity and purity were assessed using an N50 spectrophotometer. Reverse transcription was performed using a reverse transcription system. The levels of target mRNA were quantified by real-time PCR using an Applied Biosystems QuantStudio 1, with GAPDH as an internal reference gene; see Figure 4 A, We used siRNA transfection technology to knock down PBK in BV-2 cells. After transfection, the mRNA levels of IL-6, IL-1α, IL-1β, and iNOS were significantly downregulated.

[0120] 8) Flow cytometry:

[0121] siPBK and siCtrl were designed and synthesized by Sangon Biotech (Shanghai) Co., Ltd. and transiently transfected into cells according to the instructions of the transfection reagent manufacturer. The transfected cell pellets were collected, fixed with 4% paraformaldehyde solution, permeabilized with 0.1% Triton X-100, blocked with 5% BSA for 90 minutes, and incubated with the primary antibody IL-6. Subsequently, the cells were incubated with fluorescent dye-labeled secondary antibodies, washed with PBS, filtered and then passed through a CytoFlex flow cytometer (Beckman, USA). The positive cell population was analyzed; flow cytometry results showed that the level of inflammatory factor IL-6 was reduced after knocking down PBK. See Figure 4 B.

[0122] 9) Immunofluorescence:

[0123] BV-2 cells were seeded into twelve-well plates (DMEM medium containing 10% fetal bovine serum). siPBK and siCtrl were designed and synthesized by Sangon Biotech (Shanghai) Co., Ltd. and transiently transfected into the cells according to the instructions of the transfection reagent manufacturer. AB was added at a final concentration of 20 μM 36 hours after transfection. After AB acted for 1 hour, LPS was added at a final concentration of 500 ng / mL. BV-2 cells were immunofluorescently stained 4 hours later. BV-2 cells were fixed with a 4% paraformaldehyde solution at room temperature for 10 minutes, washed three times with phosphate buffered saline (PBS), permeabilized with 0.1% TritonX-100 for 10 minutes, blocked with 5% BSA at room temperature for 90 minutes, and then the corresponding primary antibodies COX-2 and P62 were added and incubated overnight at 4°C. The next day, the cells were washed three times with PBS, incubated with secondary antibodies labeled with fluorescent dyes of corresponding properties and DAPI staining at room temperature, and the sections were sealed with anti-fluorescence quenching sealing agents. The sections were observed by a Leica DM6B (Leica, Germany) microscope; see Figure 4 C, AB showed no significant effect on the expression of COX-2 and p62 in PBK knockdown BV-2 cells under LPS stimulation.

[0124] 10) Western Blot:

[0125] Western and IP cell lysis buffer (Biyuntian Biotechnology Co., Ltd.) was used to lyse BV-2 cell pellets to extract total cell protein, and the protein concentration was determined using a BCA protein concentration assay kit (Biyuntian Biotechnology Co., Ltd.). Equal amounts of protein were separated by 10% SDS-PAGE gel electrophoresis and transferred to a PVDF membrane, blocked with 5% skim milk at room temperature for 2 h, and the antibody was diluted with 5% BSA as a diluent and incubated with the primary antibody at 4°C overnight. After washing three times with Tris-HCl buffered saline (TBST) containing 1‰ Tween-20, the secondary antibody of the corresponding resistance was incubated at room temperature for 2 h, washed three times with TBST, and developed with enhanced chemiluminescence (ECL); see Figure 4 D, AB showed no significant effect on the activation of MAPK and AMPK signaling pathways in PBK knockdown BV-2 cells under LPS stimulation, as well as mitochondrial fusion protein Mfn1 and fission protein Drp1.

[0126] 11) Statistical analysis

[0127] The data were analyzed using GraphPad Prism 8 (GraphPad Software Inc, San Diego, CA, USA). The data are shown as mean ± standard error of the mean, and one-way analysis of variance was used for statistical analysis. P < 0.05 was considered statistically significant.

[0128] (3) Experimental results

[0129] Taken together, these results indicate that AB directly binds to PBK and that the protective function of AB is dependent on PBK.

[0130] Obviously, the above embodiments are only illustrative examples and do not limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. Such changes and modifications are still within the scope of protection of the present invention and do not need to exhaust all implementation methods. Obvious changes or modifications derived therefrom should also be regarded as the content of the present invention.

[0131] The present invention can also prepare common pharmaceutical preparations such as tablets, capsules, granules, etc. by conventional technical means by combining AB with a pharmaceutically acceptable carrier. Experiments have shown that pharmaceutical preparations containing AB have the efficacy of preventing and treating neuroinflammation and can be used for the preparation of drugs for preventing and treating neuroinflammation.

Claims

1. Application of 1-O-acetyl-4R,6S-inula lactone in the preparation of drugs for preventing and treating neuroinflammation.

2. Use of a pharmaceutical preparation containing 1-O-acetyl-4R,6S-inula lactone in the preparation of drugs for preventing and treating neuroinflammation.

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