Application of alkaloid DHE as TREM2 activator in preparation of medicine for treating neuroinflammation
By using alkaloid DHE as a TREM2 activator, it directly targets and activates the TREM2 receptor, solving the problem of inability to reverse neuroinflammatory and lack of targeted TREM2 drugs in the prior art, achieving significant anti-neuroinflammatory effects and safety.
Patent Information
- Application Number
- CN202411269293.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-11
- Publication Date
- 2024-12-27
AI Technical Summary
Existing drugs for treating neuroinflammatory diseases cannot reverse the condition, and no approved drugs targeting TREM2 can be used in clinical practice, which cannot effectively solve the root causes of neuroinflammatory diseases.
Using alkaloid DHE as a TREM2 activator, through strong binding activity and dose-dependent interaction with TREM2 protein, it directly targets and activates TREM2 receptors, inhibits the production of inflammatory factors of microglia, and is then used to treat and prevent neuroinflammatory diseases. .
Alkaloid DHE significantly inhibits the expression of inflammatory factors, improves motor defects induced by lipopolysaccharides, reduces the expression of inflammatory factors in the brain, has strong anti-neuroinflammatory activity and no obvious side effects are found, providing a new treatment for neuroinflammatory diseases. method.
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Abstract
Description
Technical Field
[0001] The present application relates to the field of biotechnology, and in particular to the use of an alkaloid DHE as a TREM2 activator in the preparation of a drug for treating neuroinflammation. Background Art
[0002] Neuroinflammation is a type of neurological disease whose pathogenesis is closely related to inflammatory response. Persistent neuroinflammation is closely related to common neurodegenerative diseases including multiple sclerosis (MS), Alzheimer's disease (AD), Parkinson's disease (PD), polio, etc. In the pathogenesis of neuroinflammation, the immune system and inflammatory mediators play an important role. Immune cells (such as microglia) release inflammatory mediators such as cytokines (such as tumor necrosis factor-α (TNFα), interleukin-1β (IL-1β), etc.) and chemokines (such as chemokine protein-1) after abnormal activation and damage. These mediators can cause inflammatory response and cell damage, leading to further development of neuroinflammation. At present, the drugs for treating neuroinflammation on the market can only improve symptoms, but cannot reverse the condition, so it is very important and urgent to find a new drug for treating neuroinflammation.
[0003] TREM2 (Triggering Receptor Expressed on Myeloid Cells 2) is a cell membrane receptor that is involved in regulating inflammatory responses and immune functions. TREM2 is highly expressed in microglia, and it can participate in the regulation of neuroinflammation by regulating the activation state and function of microglia. During neuroinflammation, TREM2 can induce morphological and functional changes in microglia, turning them to a state with anti-inflammatory and repair functions. Therefore, TREM2 is of great significance in the regulation of neuroinflammation. Studies have shown that the activation of the TREM2 signaling pathway can inhibit the release of inflammatory mediators, reduce the degree of activation of microglia, and thus reduce neural damage caused by inflammatory responses. TREM2 activates downstream signaling pathways, including DNAX activating protein 12 (DAP12) and TYRO protein kinase domain (TYROBP), by binding to its ligand. The activation of these signaling pathways can regulate the phagocytosis of microglia, remove neurotoxic substances, and maintain the homeostasis of the neural environment. Moreover, the TREM2 signaling pathway can also affect the production of cytokines and the transition of cell activation states in microglia, thereby affecting the process of neuroinflammation. Although some small molecule TREM2 activators, such as VG-3927 and AL002, are undergoing clinical trials for neurodegenerative diseases, there is currently no approved TREM2 drug for clinical use.
[0004] Experiments and practice have shown that the herbal medicine Ervatamia officinalis has good anti-inflammatory effects. Ervatamia officinalis is mainly distributed in the subtropical regions of China, India and South Africa. In China, it can be used to treat abdominal pain and sore throat; in India, people apply its juice to wounds to play a bactericidal and anti-inflammatory role. Ervatamia officinalis is rich in alkaloids, mainly including iboga alkaloids, corynantheine alkaloids, etc.; in South Africa, people get euphoria by chewing its leaves. In clinical practice, iboga alkaloids are used to treat opioid dependence and different inflammatory conditions. At present, there is little research on the neuroprotective effects of Ervatamia officinalis plants and their compounds. It has great development potential and is an important source of new anti-inflammatory and neuroprotective drugs. Summary of the invention
[0005] The purpose of the present application is to overcome the deficiencies of the above-mentioned prior art and to provide an application of an alkaloid DHE as a TREM2 activator in the preparation of a drug for treating neuroinflammation. The present application provides a new method for treating neuroinflammation by targeting TREM2.
[0006] To achieve the above purpose, the technical solution adopted by this application is:
[0007] The present application provides an application of an alkaloid DHE in the preparation of a TREM2 activator, wherein the structural formula of the alkaloid DHE is shown in (I);
[0008]
[0009] Currently, there is no drug on the market that can completely treat neuroinflammation without side effects, and no drug targeting TREM2 has been approved for marketing.
[0010] The present application calculated that the alkaloid DHE and TREM2 protein have strong binding activity (affinity: -7.33±0.01kcal / mol) through molecular docking experiments. The alkaloid DHE may bind to the TREM2 protein through Arg 77 and Arg 47 The amino group of DHE forms a carbon-hydrogen bond to target TREM2, and the cell thermal shift assay shows that the alkaloid DHE has the ability to directly interact with TREM2 in a dose-dependent manner.
[0011] In the technical solution of the present application, the present application discovered that the alkaloid DHE of the above structural formula is a typical corynantheine alkaloid in the Cynodon dactylon plant, which can target and activate TREM2 (mouse myeloid cell triggering receptor 2) and has an anti-neuroinflammatory effect.
[0012] The present application also provides the use of the above-mentioned alkaloid DHE as a TREM2 activator in the preparation of a drug for treating and / or preventing neuroinflammation, and the structural formula of the alkaloid DHE is shown in (I).
[0013] This application confirms for the first time that the alkaloid DHE in the medicinal dogtooth thorn plant has anti-neuroinflammatory effects, and discovers the first target of DHE, TREM2, confirming that it exerts strong anti-neuroinflammatory activity by activating TREM2.
[0014] The present application also provides the use of the above-mentioned alkaloid DHE as a TREM2 activator in the preparation of anti-neuroinflammatory drugs for treating microglia and zebrafish. The structural formula of the alkaloid DHE is shown in (I).
[0015] The alkaloid DHE provided in the present application can be used as a TREM2 activator, thereby achieving a strong anti-neuroinflammatory effect at the animal level and the cellular level, and no obvious side effects were found.
[0016] Experiments have shown that the alkaloid DHE can directly bind to and activate the TREM2 receptor in microglia, and DHE can inhibit the production of inflammatory factors by microglia induced by lipopolysaccharide. Accordingly, it can be used to protect microglia induced by lipopolysaccharide.
[0017] In in vivo studies, DHE can effectively improve motor deficits caused by LPS-induced neuroinflammation in zebrafish and reduce the expression of inflammatory factors in the brain.
[0018] As a preferred embodiment of the application described in the present application, the dosage range of the alkaloid DHE is 6 μM to 25 μM.
[0019] No obvious side effects were found when the alkaloid DHE was used within the above dosage range, and the drug had good biological activity, so the alkaloid DHE has great therapeutic potential for neurological diseases related to neuroinflammation and is worthy of further development.
[0020] As a preferred embodiment of the application described in the present application, the alkaloid DHE is used to protect microglia and zebrafish induced by lipopolysaccharide.
[0021] In some specific embodiments, the microglial cells include microglial cell line BV-2 cells.
[0022] As a preferred embodiment of the application described in the present application, the alkaloid DHE is used to inhibit the inflammatory factors induced by lipopolysaccharide.
[0023] As a preferred embodiment of the application described in the present application, the inflammatory factor includes at least one of TNF-α, IL-6, IL-10 and IL-1β.
[0024] The experiments showed that the alkaloid DHE inhibited the expression of inflammatory factors induced by LPS in microglia by activating TREM2. Specifically, the alkaloid DHE inhibited the production of TNF-α, IL-6 and IL-10 induced by LPS in microglia; the alkaloid DHE inhibited the expression of mRNA of TNF-α and IL-6 induced by LPS in microglia.
[0025] The alkaloid DHE reduced the mRNA expression levels of TNF-α, IL-6 and IL-1β induced by LPS in the zebrafish brain; the alkaloid DHE reduced the number and aggregation of neutrophils induced by LPS in the zebrafish brain; the alkaloid DHE reduced the level of ROS induced by LPS in zebrafish.
[0026] The alkaloid DHE increased the expression of TREM2 and DAP12 proteins in BV2 microglia and zebrafish. Moreover, after silencing the TREM2 gene in microglia, the trend of DHE significantly reducing the secretion of pro-inflammatory factors (TNF-α and IL-6) by BV2 cells induced by LPS was reversed, indicating that the alkaloid DHE exerted significant anti-neuroinflammatory activity by activating the TREM2 receptor in microglial BV2 cells.
[0027] As a preferred embodiment of the application described in the present application, the DHE is derived from Dogtooth Jasmine.
[0028] As a preferred embodiment of the application described in the present application, the drug is used for subcutaneous injection, intravenous injection, intramuscular injection or nasal administration.
[0029] The present application also provides a method for separating and purifying the above-mentioned alkaloid DHE, comprising the following steps:
[0030] 1) taking a leaf sample of the medicinal dogtooth hyacinth and percolating it in an ethanol aqueous solution, and concentrating the filtrate under reduced pressure to obtain a crude extract;
[0031] 2) treating the crude extract with hydrochloric acid, then extracting with CHCl3, removing the CH2Cl2 layer and retaining the aqueous phase, adjusting the pH of the aqueous phase to 9-10 with aqueous ammonia, then extracting with CH2Cl2, recovering the CH2Cl2 layer, and obtaining total alkaloids;
[0032] 3) The total alkaloids obtained in step 2) are separated and purified by macroporous resin column chromatography, MCI column chromatography and high performance liquid chromatography to obtain alkaloid DHE.
[0033] Compared with the prior art, this application has the following beneficial effects:
[0034] The present application provides an application of an alkaloid DHE as a TREM2 activator in the preparation of a drug for treating neuroinflammation. The alkaloid DHE can directly bind to and activate the TREM2 receptor, thereby inhibiting the production of inflammatory factors by microglia induced by lipopolysaccharide. Accordingly, it can be used to protect microglia induced by lipopolysaccharide. In in vivo studies, DHE can effectively improve motor defects induced by lipopolysaccharide and reduce the expression of inflammatory factors in the brain. BRIEF DESCRIPTION OF THE DRAWINGS
[0035] Figure 1 is the hydrogen nuclear magnetic resonance spectrum of DHE (400MHz, DMSO-d6);
[0036] Figure 2 DHE directly binds to TREM2 ( Figure 2 A is the molecular structure of DHE compound; Figure 2 B to Figure 2 F is the thermal stability of TREM2 and DHE (6 μM, 12 μM, and 25 μM) in BV2 microglia at a range of temperatures from 37°C to 69°C after short-term (1 h) exposure of the drug in a cell thermal shift assay.
[0037] Figure 3 This is the result diagram of the docking of DHE and TREM2 molecules;
[0038] Figure 4 The results of DHE inhibiting the expression of LPS-induced inflammatory factors in BV2 microglia by activating TREM2 ( Figure 4 A: DHE inhibits the production of TNF-α, IL-6, and IL-10 induced by LPS; Figure 4 B: DHE inhibited the expression of TNF-α and IL-6 mRNA induced by LPS; Figure 4 C: DHE increased the expression of TREM2 protein level in BV2 microglia; Figure 4 D: DHE increased the expression of DAP12 protein level in BV2 microglia; Figure 4 E to Figure 4 F shows the expression of TREM2 and DAP12 protein levels in BV2 microglia in the presence or absence of TRME2 shRNA, respectively; Figure 4 G to Figure 4 H: Effects of DHE on the expression of TNF-α and IL-6 induced by LPS in the presence or absence of TRME2 shRNA, respectively);
[0039] Figure 5 DHE inhibits the expression of LPS-induced inflammatory factors in zebrafish by activating TREM2 ( Figure 5 A: DHE effectively improves the behavioral defects of zebrafish induced by lipopolysaccharide; Figure 5 B to Figure 5 D respectively indicated that DHE reduced the mRNA expression levels of TNF-α, IL-6, and IL-1β induced by LPS in zebrafish brain; Figure 5 E and Figure 5 E': DHE reduced the number and accumulation of neutrophils induced by LPS in the zebrafish brain; Figure 5 F and Figure 5 F': DHE reduced the level of ROS induced by lipopolysaccharide in zebrafish; Figure 5 G to Figure 5 H: DHE increased the protein expression of TREM2 and DAP12 in the zebrafish brain. DETAILED DESCRIPTION
[0040] In order to better illustrate the purpose, technical solutions and advantages of the present application, the present application will be further described below in conjunction with the accompanying drawings and specific embodiments.
[0041] In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified, and the components and raw materials used in each parallel experiment are of the same type.
[0042] The experimental animals used in this application are zebrafish fry 4 days after fertilization.
[0043] The ethics number for animal experiments is UMARE-030-2017. Adult zebrafish were maintained at 28.5°C with a 14h:10h light / dark cycle. Embryos and larvae were cultured in embryo medium (0.54mM KCl, 13.7mM NaCl, 0.044mM KH2PO4, 0.025mM Na2HPO4, 0.1mM MgSO4, 0.13mM CaCl2, and 42μM NaHCO3; pH 7.4) at 28.5°C.
[0044] Example 1: Isolation and purification to obtain alkaloid DHE
[0045] This embodiment provides a method for separating and purifying the alkaloid DHE, comprising the following steps:
[0046] 10.0 kg of dried medicinal dogtooth flower branches and leaves were accurately weighed, and 95% ethanol was used for percolation four times at room temperature. The percolate was concentrated under reduced pressure to obtain a crude extract. The crude extract was dispersed in 4L 0.5% HCl water, and the pH was adjusted to 2-3. Then, an equal volume of dichloromethane (CH2Cl2) was used for extraction once, and the CH2Cl2 layer was removed to retain the aqueous phase. The aqueous phase was adjusted to pH 9-10 with ammonia water, and then extracted twice with CH2Cl2. The CH2Cl2 layer was recovered to obtain total alkaloids (39.1 g). The total alkaloids were separated by macroporous resin column chromatography (D101), and five parts (Fr.A-Fr.E) were obtained using an ethanol / water gradient elution system (0:100-95:5). The Fr.E fraction was separated by MCI column chromatography (methanol-water, 20:80-100:0) to obtain 12 parts (Fr.E1-Fr.E12). Fr.E5 was further separated by Sephadex LH-20 column chromatography (methanol) to obtain three parts (Fr.E5-1-Fr.E5-3). Fr.E5-3 was purified by preparative high performance liquid chromatography (pHPLC) using acetonitrile-water-triethylamine (70:30:0.001) to obtain DHE.
[0047] result:
[0048] The hydrogen NMR spectrum of DHE (400MHz, DMSO-d6) is as follows Figure 1 As shown; the results show that the nuclear magnetic resonance hydrogen spectrum of DHE has chemical shift, which is consistent with the molecular structure of DHE.
[0049] Example 2: Study on direct targeting of DHE to TREM2
[0050] 1. Cell thermal shift experiment:
[0051] After 1 hour of treatment with DHE or DMSO, BV2 microglia were washed, resuspended in ice PBS, and evenly transferred to PCR tubes. They were heated at different temperatures (37, 41, 45, 49, 53, 57, 61, 65, 69°C) for 3 min, and then cooled at room temperature for 3 min. The heated lysate was centrifuged at 20,000 g for 25 min at 4°C to separate soluble proteins from the precipitate, and the quantification was detected by Western blot analysis.
[0052] 2. Molecular docking experiment:
[0053] The crystal structure of TREM2 (PDB ID: 5ELI) was downloaded from Protein Data Bank (https: / / www.rcsb.org / ). The two-dimensional structure of the syn-kubin was generated using ChemDraw 20.0, and then converted into a three-dimensional structure and optimized using Chem3D 20.0. Three molecular dockings were performed using AutoDock v1.2.3 software. After docking, the best binding pose was visualized and analyzed using PyMOL (v2.3.0) and Discovery Studio (v21.1.0).
[0054] result:
[0055] Figure 2 DHE directly binds to TREM2. Figure 2 A is the molecular structure of DHE compound; Figure 2 B to Figure 2 F is the thermal stability of TREM2 in BV2 microglia cells exposed to different concentrations of DHE (6 μM, 12 μM and 25 μM) at a temperature range from 37°C to 69°C for a short time (1 h) in a cell thermal shift assay.
[0056] Figure 3 The results of molecular docking of DHE and TREM2.
[0057] DHE-TREM2 binding was confirmed by cellular thermal shift assay (CETSA) in BV2 microglia, which detects changes in the protein melting curve caused by drug binding and changes in the shift of protein melting temperature (Tm) to judge whether the molecule binds to the target protein.
[0058] The results showed that within the temperature range of 37°C to 69°C, compared with the DMSO solvent control group, the addition of 6 μM DHE slightly increased the soluble protein accumulation of TREM2, while 12 μM and 25 μM DHE significantly increased the soluble protein accumulation of TREM2 and shifted the Tm value of TREM2 by 9.2°C and 9.5°C, respectively, indicating that DHE has the ability to directly interact with TREM2 in a dose-dependent manner.
[0059] Figure 3 The results further showed that the molecular docking experiment calculated that there was a strong binding activity between DHE and TREM2 protein (affinity: -7.33±0.01kcal / mol), and DHE may bind to Arg 77 and Arg 47 The amino group of the protein forms a carbon-hydrogen bond to target and bind TREM2.
[0060] Example 3. Test of the anti-neuroinflammatory activity of DHE in microglia by activating TREM2
[0061] The immortalized microglial cell line BV-2 was obtained from the Kunming Cell Bank of Kunming Institute of Zoology. The cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% PS (v / v) at 37°C in a 5% CO2 cell culture incubator. 10% heat-inactivated fetal bovine serum and 1% PS (v / v) were added to BV2 cells. The cells were pretreated with 6μM, 12μM and 25μM DHE for 1 hour before lipopolysaccharide (800ng / ml) stimulation. The experimental data are expressed as Mean±SD. All experimental data were statistically analyzed using GraphPad Prism 8 software. The parameters between groups were tested using ANOVA, and P<0.05 was considered statistically significant.
[0062] 1. Enzyme-linked immunosorbent assay (ELISA):
[0063] BV-2 cells in the logarithmic growth phase were digested and counted, and 1×10 5 Each well was inoculated into a 24-well plate. After culturing for 24 hours, different concentrations of DHE (6μM, 12μM and 25μM) were added for pretreatment for one hour, followed by the addition of 800ng / ml LPS for further culturing for 24 hours (the blank group was not added with LPS). The culture supernatant was collected after treatment. The levels of inflammatory mediators TNF-α, IL-6 and IL-10 were detected using ELISA kits.
[0064] 2. Extraction of total cell RNA and reverse transcription polymerase chain reaction (RT-PCR):
[0065] Total RNA from drug-treated BV2 microglia was extracted using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. RNA was reverse transcribed into cDNA using Evo M-MLV reverse transcription reagent (Accurate Biology) with random primers according to the manufacturer's protocol. RT-PCR was performed using Premix Ex Taq TM II kit and Mx3005P qPCR system (Agilent Technologies, Santa Clara, CA, USA). β-actin was used as the internal reference gene. The primer sequences of each gene (as shown in SEQ ID NO: 1 to SEQ ID NO: 6) are shown in Table 1.
[0066] Table 1 Primers used for quantitative RT-PCR in BV2 microglial cell samples
[0067]
[0068] 3. TREM2 shRNA transfection experiment:
[0069] A lentiviral vector expressing mouse TREM2-shRNA was constructed. The shRNA sequence targeting TREM2 was 5'-GGAATCAAGAGACCTCCTTCC-3' (SEQ ID NO: 7). The scrambled RNA sequence used as a control was 5'-TTCTCCGAACGTGTCACGT-3' (SEQ ID NO: 8). The shRNA was transfected into BV2 microglia cells using 8000 reagent (Invitrogen) was cultured to 60-70% confluence according to the manufacturer's protocol. 48 h after transfection, they were divided into six groups: Control shRNA group, Control shRNA+LPS group, Control shRNA+LPS+DHE (25 μM) group, TREM2 shRNA group, TREM2 shRNA+LPS group, and TREM2 shRNA+LPS+DHE (25 μM) group. The expression level of TREM2 protein was detected by Western Blot experiment.
[0070] 4. Western Blot Detection:
[0071] BV2 cells were collected at 3×10 5100 μg / well were inoculated into 10 cm culture dishes. After culturing for 24 h, different concentrations of DHE (6 μM, 12 μM and 25 μM) were added for pretreatment for one hour, and then 800 ng / ml of LPS was added for further culturing for 24 h (no LPS was added to the blank group). The cell supernatant was aspirated, washed twice with PBS, and then lysed with RIPA buffer containing 1% saturated PMSF and 1% protease and phosphatase inhibitor mixture. The protein concentration was determined using Bradford Protein Assay Kit (ThermoFisherScientific, Waltham, MA, USA). Protein samples were collected and the expression levels of TREM2 and DAP12 proteins were detected by Western Blot, with β-actin as an internal reference.
[0072] result:
[0073] Figure 4 DHE inhibits the expression of LPS-induced inflammatory factors in BV2 microglia by activating TREM2. Figure 4 A: DHE inhibits the production of TNF-α, IL-6, and IL-10 induced by LPS; Figure 4 B: DHE inhibited the expression of TNF-α and IL-6 mRNA induced by LPS; Figure 4 C: DHE increased the expression of TREM2 protein level in BV2 microglia; Figure 4 D: DHE increased the expression of DAP12 protein level in BV2 microglia; Figure 4 E to Figure 4 F shows the expression of TREM2 and DAP12 protein levels in BV2 microglia in the presence or absence of TRME2 shRNA, respectively; Figure 4 G to Figure 4 H shows the effect of DHE on the expression of TNF-α and IL-6 induced by LPS in the presence or absence of TRME2 shRNA.
[0074] from Figure 4 A The results show that the secretion of TNF-α and IL-6 in the control group BV2 cells is very low. Compared with the control group, after LPS (800ng / ml) induced BV2 cells, the release of the two can be significantly increased, and the release of IL-10 is significantly reduced, indicating that LPS activates microglia and promotes microglia polarization to M1 type, producing an inflammatory response. Compared with the LPS group, DHE can significantly reduce the release of TNF-α and IL-6, and increase the expression of IL-10, and it is concentration-dependent, indicating that DHE has a significant inhibitory effect on LPS-induced BV2 cells to produce pro-inflammatory factors, and has a significant promoting effect on anti-inflammatory factors. Figure 4B. The results show that DHE can significantly reduce the expression levels of TNF-α and IL-6 mRNA induced by LPS.
[0075] Figure 4 C to Figure 4 D The results showed that compared with the control group, the LPS group did not significantly increase the protein expression levels of TREM2 and DAP12, but 25 μM DHE significantly increased their levels.
[0076] Figure 4 E to Figure 4 F shows that after silencing the TREM2 gene in BV2 cells with TREM2 shRNA, the effect of DHE on increasing the levels of TREM2 and DAP12 proteins was eliminated; in addition, Figure 4 G to Figure 4 H is that after silencing the TREM2 gene in microglia, DHE reversed the trend of significantly reducing the secretion of pro-inflammatory factors (TNF-α and IL-6) by BV2 cells induced by LPS, indicating that DHE exerts significant anti-neuroinflammatory activity by activating TREM2 receptors in microglial BV2 cells.
[0077] Example 4. Anti-neuroinflammatory activity test of DHE in zebrafish by activating TREM2
[0078] Zebrafish larvae at 4 days post fertilization (4-dpf) were randomly divided into 6 groups, namely: normal control group (Control), LPS group (LPS, 2.5kg / ml), DHE (25μM) + LPS group, DHE group (25μM), at least 12 in each group, and placed in 6-well plates. The control group and LPS group were pretreated with 0.1% DMSO embryo culture medium for 24h, while the drug-treated group was pretreated with the corresponding concentration of DHE drug for 24h; at 5-dpf (5 days after fertilization), 0.02% w / v tricaine was used for anesthesia, and LPS was microinjected into the brain of zebrafish in the LPS group and DHE (25μM) + LPS group, 2nl per fish, while the control group and the DHE group without LPS were injected with the same volume of sterile PBS. Behavioral, Western blot analysis and QT-PCR analysis experiments were performed 24h later. The experimental data were expressed as Mean ± SEM. All experimental data were statistically analyzed using GraphPad Prism 8 software. The inter-group parameters of zebrafish in each group were tested using ANOVA, and P < 0.05 was considered statistically significant.
[0079] 1. Motor behavior test:
[0080] After treatment with DHE and LPS, 6-dpf (6 days after fertilization) zebrafish larvae were transferred to 96-well plates (1 fish / well). Zebrafish movement was measured for 30 minutes using a zebrafish tracking system (Viewpoint Life Sciences, Lyon, France). The total travel distance of the zebrafish larvae was also calculated. Since direct injection of LPS into the brain can cause inflammation of brain tissue, resulting in brain nerve damage, thereby reducing motor ability. Therefore, this experiment examined whether DHE had an improvement effect on LPS-induced zebrafish motor defects by judging the motor ability of zebrafish larvae.
[0081] 2. Zebrafish neutrophil test:
[0082] After the 4-day post-fertilization Tg (Mpo: EGFP) zebrafish larvae were treated with the above-described drugs, the 5-dpf larvae were microinjected with LPS or PBS into the brain. The 6-dpf larvae were subjected to brain live imaging under Leica M205FA fluorescence stereo microscopes to observe the number and aggregation of neutrophils. The obtained images were processed and analyzed using ImageJ software. Since direct injection of LPS into the brain will lead to a strong immune response in the brain, the number of immune cells such as neutrophils and microglia will increase and their aggregation will be activated. Therefore, this experiment observed the number and aggregation of immune cells represented by neutrophils in the brain of zebrafish larvae to judge whether DHE can reduce the number and aggregation of LPS-induced immune cells.
[0083] 3. ROS test:
[0084] After 4-day postfertilization AB zebrafish larvae were treated with the above drugs and LPS, 6-dpf larvae were immersed in DCFH-DA (10 μM) solution and incubated for 30 min in the dark at 28.5°C. After washing three times with culture medium, the larvae were observed with a fluorescent stereo microscope, and the images were analyzed with ImageJ software.
[0085] 4. Extraction of total RNA from zebrafish brain and reverse transcription polymerase chain reaction (RT-PCR):
[0086] For 6-dpf zebrafish samples, 0.02% w / v tricaine was used for anesthesia, and the head parts of zebrafish larvae without eyes and yolk sac were collected (30 per group). Total RNA from these zebrafish tissues was extracted using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. RNA was reverse transcribed into cDNA using Evo M-MLV reverse transcription reagent (Accurate Biology) with random primers according to the manufacturer's protocol. RT-PCR detection was performed using Premix Ex Taq TM II kit and Mx3005P qPCR system (Agilent Technologies, Santa Clara, CA, USA). ef1a was used as the internal reference gene. The primer sequences of each gene (SEQ ID NO: 9 to SEQ ID NO: 16) are shown in Table 2.
[0087] Table 2 Primers used for quantitative RT-PCR of zebrafish samples
[0088]
[0089] 5. Total protein extraction and Western Blot detection from zebrafish brain:
[0090] For 6-dpf zebrafish samples, 0.02% w / v tricaine was used for anesthesia, and the head parts of zebrafish larvae without eyes and yolk sac were collected (30 per group). Cells were lysed with RIPA buffer containing 1% saturated PMSF and 1% protease and phosphatase inhibitor cocktail. Protein concentration was determined using Bradford Protein Assay Kit (ThermoFisher Scientific, Waltham, MA, USA). Protein samples were collected and Western Blot was used to detect the expression levels of IBA1 and TREM2 proteins, with β-actin as an internal reference.
[0091] result:
[0092] Figure 5 The figure shows that DHE inhibits the expression of LPS-induced inflammatory factors in zebrafish by activating TREM2. Figure 5 A: DHE effectively improves the behavioral defects of zebrafish induced by lipopolysaccharide; Figure 5 B to Figure 5 D: DHE reduced the mRNA expression levels of TNF-α, IL-6, and IL-1β induced by LPS in zebrafish brain; Figure 5 E to Figure 5 E': DHE reduced the number and accumulation of neutrophils induced by LPS in the zebrafish brain; Figure 5 F to Figure 5 F': DHE reduced the level of ROS induced by lipopolysaccharide in zebrafish; Figure 5 G to Figure 5 H respectively showed that DHE increased the protein expression levels of TREM2 and DAP12 in the zebrafish brain.
[0093] Figure 5A: LPS significantly reduced the total swimming distance of zebrafish, while DHE (25 μM) + LPS group treatment significantly increased the swimming distance of zebrafish. This indicates that DHE can restore the motor behavior defects of zebrafish larvae induced by LPS. Under normal circumstances, microglia are in a "resting" state, while in pathological conditions, microglia are widely activated and produce a large number of inflammatory factors to damage neurons. Figure 5 B to Figure 5 D: The DHE group significantly reduced the LPS-induced increase in the mRNA expression levels of TNF-α, IL-6 and IL-1β, indicating that DHE has a significant inhibitory effect on LPS-induced neuroinflammation. Figure 5 E to Figure 5 E' are the analysis and distribution diagrams of the number of neutrophils in the brain of zebrafish larvae, respectively. The results show that compared with the control group, the LPS group significantly increased the number and aggregation of neutrophils in the zebrafish brain, indicating that brain injection of LPS caused a strong neuroimmune response.
[0094] After administration of DHE, the number of neutrophils decreased, further verifying the effect of DHE in inhibiting neuroinflammation, while the group given only high-dose DHE did not cause a significant increase in the number and aggregation of neutrophils. Figure 5 F-F' shows that the DHE group significantly reduced the production of LPS-induced ROS in zebrafish larvae, reflecting the certain antioxidant capacity of DHE. Figure 5 GH and high-dose DHE significantly increased the expression of TREM2 and DAP12 proteins in the zebrafish brain, indicating that DHE effectively inhibits the occurrence and development of neuroinflammation by activating TREM2 receptors.
[0095] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present application rather than to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, ordinary technicians in this field should understand that the technical solution of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present application.
Claims
1. The use of alkaloid DHE in the preparation of TREM2 activator, characterized in that: The structural formula of the alkaloid DHE is shown in (I); 2. Use of alkaloid DHE as a TREM2 activator in the preparation of a drug for treating and / or preventing neuroinflammation, characterized in that: The structural formula of the alkaloid DHE is shown in (I); 3. Use of alkaloid DHE as a TREM2 activator in the preparation of an anti-neuroinflammatory drug for treating microglia and zebrafish, characterized in that: The structural formula of the alkaloid DHE is shown in (I); 4. The use according to claim 2 or 3, characterized in that The dosage range of the alkaloid DHE is 6 μM to 25 μM.
5. The use according to claim 3, characterized in that The alkaloid DHE protects microglia and zebrafish from lipopolysaccharide-induced damage.
6. The use according to claim 3, characterized in that The alkaloid DHE is used to inhibit the inflammatory factors induced by lipopolysaccharide.
7. The use according to claim 6, characterized in that The inflammatory factor includes at least one of TNF-α, IL-6, IL-10 and IL-1β.
8. The use according to claim 3, characterized in that The alkaloid DHE is derived from Cynanchum dactylum.
9. The use according to claim 2 or 3, characterized in that: The medicine is used for subcutaneous injection, intravenous injection, intramuscular injection or nasal administration.
10. A method for separating and purifying alkaloid DHE, characterized in that: The following steps are involved: 1) taking a leaf sample of the medicinal dogtooth hyacinth and percolating it in an ethanol aqueous solution, and concentrating the filtrate under reduced pressure to obtain a crude extract; 2) treating the crude extract with hydrochloric acid, then extracting with CHCl3, removing the CH2Cl2 layer and retaining the aqueous phase, adjusting the pH of the aqueous phase to 9-10 with aqueous ammonia, then extracting with CH2Cl2, recovering the CH2Cl2 layer, and obtaining total alkaloids; 3) The total alkaloids obtained in step 2) are separated and purified by macroporous resin column chromatography, MCI column chromatography and high performance liquid chromatography to obtain alkaloid DHE.