Application of sclareol in the preparation of drugs for inhibiting microglia-mediated inflammation

Abstract

The present invention provides the use of sclareol in the preparation of drugs for inhibiting microglia-mediated inflammation, and belongs to the field of biomedicine technology. The present invention observes inflammatory response indicators through animal intraperitoneal injection of LPS model, BV2 cell LPS stimulation model and substantia nigra α-Syn overexpression PD model mice, and finds that sclareol can inhibit the inflammatory response caused by central microglia, improve the pathological manifestations of Parkinson's disease, and provide a new approach for the treatment of degenerative diseases of the central nervous system.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to the application of sclareol in the preparation of a drug for inhibiting microglia-mediated inflammation. Background Art

[0002] Inflammatory responses in the central nervous system are closely related to many neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease (PD). Microglia, as the main immune cells in the central nervous system, play a major role in inflammatory responses in the central nervous system. Sclareol is derived from the inflorescence of the plant sclareol and the leaves of tobacco. Studies have shown that sclareol has anti-tumor, immunomodulatory, and antioxidant effects. However, there have been no studies on whether sclareol can inhibit the inflammatory response mediated by central microglia. Summary of the invention

[0003] In view of this, the present invention aims to provide the use of sclareol in the preparation of drugs for inhibiting microglia-mediated inflammation. The sclareol of the present invention can inhibit the central inflammatory response of microglia, providing a possible new approach for the treatment of degenerative diseases of the central nervous system.

[0004] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0005] The present invention provides application of sclareol in preparing a drug for inhibiting microglia-mediated inflammation.

[0006] Preferably, the sclareol can improve the hypoactivity, memory loss and motor impairment caused by brain inflammation in mice.

[0007] Preferably, the sclareol reduces the increase of inflammatory factors in the mouse cortex and downregulates inflammation-related pathways.

[0008] Preferably, the sclareol inhibits the activation of mouse cortical microglia.

[0009] Preferably, the sclareol reduces the level of inflammatory factors released by BV2 cells induced by LPS stimulation and inhibits the level of oxidative stress of BV2 cells induced by LPS stimulation.

[0010] The present invention also provides the use of sclareol in preparing drugs for preventing and / or treating Parkinson's disease.

[0011] Preferably, the sclareol improves PD-related movement disorders in mice and activation of microglia in the substantia nigra of mice caused by overexpression of α-Syn in the substantia nigra.

[0012] Preferably, the sclareol improves the TH-positive neuron damage in the substantia nigra of mice caused by overexpression of α-Syn in the substantia nigra, the level of inflammatory factors in the substantia nigra of mice with PD, and the accumulation of α-Syn.

[0013] Preferably, the usage amount of sclareol is 35-65 mg / kg.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The present invention proposes for the first time the use of sclareol in inhibiting microglia-mediated inflammation. Experimental studies have found that in the neuroinflammation model of intraperitoneal injection of LPS, the sclareol can improve the hypoactivity, memory loss, and motor impairment caused by central nervous system inflammation in mice caused by LPS injection, reduce the increase of inflammatory factors in the mouse cortex, inhibit the activation of microglia in the mouse cortex, and downregulate inflammation-related pathways. In the BV2 cell model stimulated by LPS, sclareol reduces the levels of inflammatory factors and oxidative stress released by BV2 cells caused by LPS stimulation.

[0016] The present invention also proposes for the first time that in the PD model of substantia nigra α-Syn overexpression, sclareol improves PD-related pathological symptoms of mice, inhibits microglial activation and protects TH-positive neurons, and reduces the level of inflammatory factors and α-Syn accumulation in the substantia nigra, indicating that sclareol has a certain therapeutic effect on Parkinson's disease and provides a new approach for the treatment of degenerative diseases of the central nervous system. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The stereochemical formula (left) and structural formula (right) of sclareol.

[0018] Figure 2 Results of behavioral experiments on mice 24 hours after LPS injection (AC: open field test, DF: water maze test, GH: pole climbing test, n≥5, where the vehicle group in Figures B and C refers to the LPS group).

[0019] Figure 3 Expression of inflammatory factors in the mouse cortex 24 hours after LPS injection (A: mRNA level of inflammatory factors, BD: protein level of inflammatory factors, n≥5).

[0020] Figure 4 Activation of cortical microglia in mice 24 hours after LPS injection (A: immunofluorescence staining, scale bars: 2 mm and 20 μm; BD: statistical results of immunofluorescence staining, n=3).

[0021] Figure 5RNA-Seq analysis results (A: Venn diagram, B: GSEA analysis results, C: GO analysis results, D: KEGG analysis results, n=3).

[0022] Figure 6 The results of the effects of sclareol on BV2 cell viability and NO release (AB: the effect of sclareol concentration on BV2 cell viability, C: the changes in NO release with increasing sclareol concentrations after BV2 cells were stimulated with LPS for 24 hours, n=3).

[0023] Figure 7 Expression of inflammatory factors in BV2 cells 24 hours after the addition of LPS (A: RNA level of inflammatory factors, BC: protein level of inflammatory factors, n=3).

[0024] Figure 8 Effects of sclareol on oxidative stress in BV2 cells stimulated by LPS 24 h after addition of LPS (AB: JC-1 staining, C: glutathione peroxidase activity detection, DE: intracellular ROS level detection, n≥3).

[0025] Fig. 9 Behavioral test results of α-Syn overexpression PD model mice (A: cylinder test, B: rotarod test, CD: climbing pole test, n>5).

[0026] Fig.10 Activation of microglia in the substantia nigra of PD model mice with α-Syn overexpression (the upper figure is the immunofluorescence staining image, scale bars: 2mm, 100μm and 25μm; the middle and lower figures are the statistical results of immunofluorescence staining images, n>3).

[0027] Fig.11 Staining results of TH-positive neurons in the substantia nigra of PD model mice with α-Syn overexpression (the upper figure is the staining image, scale bar 100μm; the lower figure is the corresponding statistical results of the staining image, n>3).

[0028] Fig.12 Levels of inflammatory factors in the substantia nigra of PD model mice overexpressing α-Syn (the upper figure is the immunoblot, the lower figure is the statistical results, n≥4). DETAILED DESCRIPTION

[0029] The present invention provides the use of sclareol in the preparation of a drug for inhibiting microglia-mediated inflammation. The sclareol structural formula and stereochemical structural formula of the present invention are as follows: Figure 1 The sclareol of the present invention can be obtained from commercial channels well known in the art, or can be obtained by a preparation method well known to those skilled in the art.

[0030] In the present invention, the sclareol can improve the hypoactivity, memory loss, and motor ability loss caused by LPS injection-induced brain inflammation in mice, the sclareol reduces the increase of inflammatory factors in the mouse cortex caused by LPS injection, the sclareol inhibits the activation of microglia in the mouse cortex, and the sclareol downregulates inflammation-related expression pathways. The present invention intraperitoneally injects sclareol into mice 5 to 9 days after intraperitoneal injection of LPS, and observes the behavior of the mice. In the mouse model experiment of the present invention, the amount of sclareol used is 35 to 65 mg / kg, preferably 50 mg / kg. The administration methods of the sclareol of the present invention include oral, intravenous, intramuscular, subcutaneous, nasal, oral mucosa, eyes, lungs and respiratory tract, skin, vagina, rectum, etc.

[0031] In the present invention, the sclareol reduces the level of inflammatory factors released by BV2 cells induced by LPS stimulation. In the BV2 cell model experiment of the present invention, the concentration of the sclareol used is 10-80 μM, preferably 40 μM.

[0032] The present invention also provides the use of sclareol in the preparation of drugs for the prevention and / or treatment of Parkinson's disease. The sclareol of the present invention can improve the PD-related pathological symptoms of mice with substantia nigra α-Syn overexpression, inhibit microglial activation and protect TH-positive neurons, and reduce the level of inflammatory factors and α-Syn accumulation in the substantia nigra. In the mouse PD model experiment of the present invention, sclareol was given for treatment for 2 weeks after the injection of virus for stable expression, which improved the PD-related pathological symptoms of mice with substantia nigra α-Syn overexpression. The usage amount of sclareol of the present invention is preferably 50 mg / kg. The administration methods of sclareol of the present invention include oral, intravenous, intramuscular, subcutaneous, nasal, oral mucosa, eyes, lungs and respiratory tract, skin, vagina, rectum, etc.

[0033] In the present invention, the drug includes liquid dosage forms, solid dosage forms or semisolid dosage forms. The liquid dosage forms of the present invention include solutions, emulsions, suspensions, injections, eye drops, nasal drops, lotions and liniments, etc.; solid dosage forms include tablets (including ordinary tablets, enteric-coated tablets, lozenges, dispersible tablets, chewable tablets, effervescent tablets, orally disintegrating tablets), capsules (including hard capsules, soft capsules, enteric-coated capsules), granules, powders, micropills, dropping pills, suppositories, films, patches, aerosols (powders), sprays, etc.; semisolid dosage forms include ointments, gels, pastes, etc.

[0034] In the present invention, unless otherwise specified, all components or reagents are commercially available products well known to those skilled in the art.

[0035] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] The antibodies required in the experiment of the embodiment of the present invention include: anti-β-actin antibody (Santa Cruz Biotechnology, USA), recombinant Anti-IL-6 antibody [EPR21710] (ab229381), recombinant Anti-iNOS antibody [EPR16635] (ab178944), recombinant Anti-Iba1 antibody [EPR16588] (ab178846), Anti-TNF alpha antibody [EPR19147] (ab183218), Anti-MCP1antibody (ab25124), recombinant Anti-Alpha-synuclein antibody [MJFR1] (ab138501), Goat Anti-Mouse IgG H&L (Alexa Fluor 488) (ab150113), Goat Anti-Rabbit IgGH&L (Alexa Fluor 594) (ab150080), Donkey Anti-Goat IgGH&L (AlexaFluor647) (ab150135) were purchased from Abcam, UK), Cox2 (D5H5) XP Rabbit mAb (12282T) (CST, USA), Anti-IL1beta antibody (Beijing Biotechnology Co., Ltd.), Anti-mouseCD16 / CD32 antibody (553141) (BD, USA), Mouse MMR / CD206 antibody (AF2535) (R&D Systems, USA), MonoclonalAnti-TH antibody (AMAB91112) (Merck, USA).

[0037] Example 1 Effect of Sclareol on LPS-induced Mouse Model

[0038] 1. Experimental grouping: 8-week-old male C57BL / 6 wild-type mice were selected for the experiment. They were divided into saline group, LPS group and drug treatment group (Scl+LPS group), with 5 mice in each group. The mice in the Saline group and LPS group were intraperitoneally injected with solvent (98% corn oil + 2% DMSO) at 4:00 pm every day, and the Scl+LPS group was injected with a certain concentration of sclareol (Tokyo Chemical Industry Co., Ltd., Japan) every day. After 7 days, the LPS group and Scl+LPS group were intraperitoneally injected with LPS 3mg / kg, and the Saline group was injected with saline. After 24h, animal behavior experiments and material collection were performed. In the open field test, the concentrations of sclareol were 25mg / kg, 50mg / kg, 75mg / Kg, and 100mg / Kg, respectively, and the concentration of sclareol in subsequent experiments was 50mg / Kg.

[0039] 2. Behavioral testing

[0040] Mice with neuroinflammation have poor motor performance, impaired memory, and may show depression. In order to detect the anti-neuroinflammatory effect of clarysol, behavioral tests were performed. In the open field test, the movement distance of mice in the center position is negatively correlated with the depressive state of the mice, and the movement distance of mice in the entire space shows the level of exercise. The climbing pole test can detect the motor coordination ability and movement speed of mice. In the water maze test, the movement distance of mice in the target quadrant is often used to detect the spatial memory ability of mice.

[0041] (1) Open field test: 24 h after LPS injection, the animals were placed in the center of an open field test apparatus with a bottom area of ​​50*50 cm. After the mice explored for 1 min, their 10-min movement trajectory was recorded and analyzed.

[0042] (2) Water maze experiment: On the first day of training, the platform was visible. The mice were placed in water from four different directions to train them to get on the platform. On days 2-6, polymer pigments were added to submerge the platform. The mice were placed in water from four different directions. The position of the platform remained unchanged so that the mice could remember the position of the platform. On the last day (24 hours after LPS injection), the platform was removed and the mice were placed in water from a new position farthest from the platform. The movement trajectory of the mice was recorded for one minute and analyzed.

[0043] (3) Pole climbing test: 24 h after LPS injection, the mice were placed head up on a 50 cm, upright hard plastic rod wrapped with gauze. The time it took for the mice to change from the head-up position to the head-down position and the total time it took to climb to the bottom of the rod were recorded (the mice had to be trained two days in advance).

[0044] Results Figure 2As shown. The results of the open field test showed that with the increase of the concentration of sclareol, the movement distance of mice in the center of the open field and the total movement distance showed a trend of first increasing and then decreasing. At a concentration of 50 mg / Kg, the mice had the best movement state and the lowest depression level, which was significantly improved compared with the LPS group, indicating that 50 mg / kg was the optimal drug concentration. The results of the pole climbing experiment showed that compared with the LPS group, the time for mice to turn their heads when climbing the pole was shortened, the climbing speed was accelerated, and the sliding score was reduced. This indicates that the mice's motor coordination ability and movement speed have been restored. In order to detect the memory impairment status of mice, a water maze experiment was conducted. The experimental results showed that the target quadrant distance of mice was greatly reduced after LPS injection, indicating that the memory of mice was reduced, and the injection of sclareol improved this symptom. In summary, sclareol improved the symptoms of motor and memory impairment caused by intraperitoneal injection of LPS.

[0045] 3. Detection of inflammatory factor levels

[0046] (1) Fresh mouse cortex / cells from each group were obtained and RNA was extracted using FastPure Cell / Tissue Total RNA Isolation Kit (Nanjing Novogene Biotech Co., Ltd.). The extraction steps were referred to the kit instructions. The extracted total RNA can be directly used for downstream experiments or stored at -70°C.

[0047] (2) Reverse transcription: The extracted RNA was reverse transcribed according to the instructions of HiScript III RTSuperMix for qPCR (+gDNAwiper) (Nanjing Novogene Biotech Co., Ltd.). The cDNA product was stored at -20°C.

[0048] (3) qPCR reaction: Prepare the reaction system according to Table 1 below, and perform qPCR reaction with the primers described in Table 3 according to the reaction conditions shown in Table 2. After the reaction, perform relative quantification according to the average Ct value obtained. The amplification data was calculated using the 2-ΔΔCT relative quantification method, and the data were processed as follows: Δct = ct (gene of interest) - ct (β-actin), ΔΔct = Δct (test group) - Δct (control), and the expression multiple of the test gene in each group relative to the test gene in the control group was 2-ΔΔct.

[0049] Table 1 qPCR reaction system

[0050]

[0051] Table 2 qPCR reaction conditions

[0052]

[0053]

[0054] Table 3 Primer sequences involved in the qPCR process

[0055]

[0056] (4) ELISA experiment: 1) Add 100 μL of standard working solution or sample to the corresponding wells and incubate at 37°C for 90 minutes. 2) After discarding the liquid in the plate, immediately add 100 μL of biotinylated antibody working solution and incubate at 37°C for 60 minutes. 3) Discard the liquid in the plate and wash the plate 3 times. 4) Add 100 μL of HRP enzyme conjugate working solution to each well, incubate at 37°C for 30 minutes, discard the liquid in the plate, and wash the plate 5 times. 5) Add 90 μL of substrate solution to each well and incubate at 37°C for about 15 minutes. 6) Add 50 μL of stop solution to each well. 7) Immediately read the data at a wavelength of 450 nm and process the data. The kits used include the reagents required for ELISA experiments: Mouse IL-6 (Interleukin 6) ELISA Kit, Mouse IL-1β (Interleukin 1Beta) ELISA Kit, Mouse TNF-α (Tumor Necrosis Factor Alpha) ELISA Kit (Wuhan Elaruite Biotechnology Co., Ltd.).

[0057] The main characteristic of neuroinflammation at the molecular level is the excessive release of inflammatory factors. Figure 3 . The results of qPCR experiments showed that 24 hours after LPS injection, the mRNA levels of inflammatory factors IL6, CCL2, COX2, TNF-α, IL-1β, iNOS, and Tlr4 in the cortex of mice in the LPS group increased significantly, and the mRNA levels of inflammatory factors in the cortex of mice in the Scl+LPS group decreased compared with the LPS group. The results of ELISA experiments showed that 24 hours after LPS injection, the levels of IL6, TNF-α, and IL-1β in the LPS group increased significantly, and the drug-treated group inhibited this increase. The above results show that, whether at the mRNA or protein level, sclareol inhibited the increase in the level of inflammatory factors in the cortex of mice stimulated by LPS, and has a neuroprotective effect of antagonizing neuroinflammation.

[0058] 4. Immunofluorescence observation of the effect of sclareol on LPS-induced mouse cortical microglia

[0059] The mouse was over-anesthetized, the mouse chest cavity was opened, the perfusion needle was inserted into the left ventricle, the right atrial appendage was cut, and the perfusion pump was immediately turned on to perform saline perfusion (about 5 minutes). After the blood was discharged from the right atrial appendage, 4% paraformaldehyde (PFA, Beijing Lanjieke Technology Co., Ltd.) was used for perfusion for 10 minutes. Subsequently, the mouse brain tissue was carefully peeled out and fixed in 4% PFA for 24 hours. After fixation, the brain tissue was dehydrated with 30% sucrose gradient. Dehydrate for 2 weeks. After dehydration, the brain tissue was embedded in OCT (SAKURA, USA) and stored in a -80°C refrigerator.

[0060] Preparation of slices: 1) Take out the embedded block from -80℃ and return it to -20℃ before placing it in a freezing microtome. 2) Adjust the position of the brain tissue and slice it, with the slice thickness set to 20μm. 3) Wash the slices in TBS and remove OCT.

[0061] Antibody incubation: 1) Place the slices in immunostaining blocking solution (Shanghai Beyotime Company) at room temperature and block for 2 hours. 2) Transfer the blocked slices to the primary antibody diluent (Shanghai Beyotime Company) and incubate overnight at 4°C on a shaker. 3) After rewarming at room temperature for 1 hour the next day, wash the slices with TBST for 5 minutes × 3 times, transfer the slices to the secondary antibody diluent (Shanghai Beyotime Company) and incubate at room temperature in the dark for 2 hours. 4) Wash the slices with TBS for 5 minutes × 3 times, and add anti-fluorescence attenuation mounting medium (containing DAPI). Cover the slices with a coverslip to avoid bubbles. Observe and photograph under a fluorescence microscope. See the results. Figure 4 .

[0062] In the state of neuroinflammation, microglia proliferate and transform into an inflammatory phenotype, and their morphology changes, which are manifested as a decrease in the number of branches, shortening, and thickening, an enlarged cell body, and an enhanced phagocytic and migratory ability. A 20× field of view of the mouse cortex in the same part was taken, and the slice thickness was 20μm. After the fluorescent image was binarized, it was skeletonized using the built-in plug-in in ImageJ, and then a skeleton analysis was performed. The obtained data was processed, and the noise was deleted with a threshold of the number of branches greater than or equal to 2 and the longest branch length greater than or equal to 0.4, and the number of branches and the average branch length were counted. The experimental results are as follows Figure 4 The experimental results showed that, per unit field of view, the number of microglia in the LPS group increased, the number of branches decreased, and the length of branches shortened. Compared with the LPS group, the number of microglia in the drug-treated group was less, the number of branches increased, and the length of branches prolonged. This suggests that sclarea alcohol inhibits the inflammatory morphological transformation of microglia and has an antagonistic effect on neuroinflammation.

[0063] 5. Transcriptome analysis: 6 hours after LPS injection, the mouse cortex was collected, 3 mice in each group, and sent to the company (Anno Gene) for transcriptome sequencing. After the sequencing results came out, GO, KEGG, and GSEA analysis were performed on genes with different expressions between the solvent group and the sclareol group.

[0064] Depend on Figure 5 As shown in the figure, the differentially expressed genes after RNA-Seq sequencing were plotted into a Venn diagram, showing that there were 1502 differentially expressed genes between the Saline group and the LPS group, 1343 differentially expressed genes between the Saline group and the Scl+LPS group, and 61 differentially expressed genes between the LPS group and the Scl+LPS group. The differentially expressed genes between the LPS group and the Scl+LPS group were subjected to GSEA enrichment analysis, and the results showed that the pathways strongly related to inflammation: JAK-STAT pathway, TNF signaling pathway, and Toll-like receptor signaling pathway were all downregulated in the Scl+LPS group, further verifying the anti-inflammatory effect of sclareol.

[0065] The differentially expressed genes between the LPS group and the Scl+LPS group were subjected to GO analysis. The results showed that in terms of biological process, these differentially expressed genes were mainly enriched in inflammation-related processes such as leukocyte infiltration, leukocyte adhesion, and leukocyte migration. In terms of cellular component, they were enriched in dopaminergic synapses, receptor complexes, and other contents. In terms of molecular function, they were enriched in glutamate receptor binding, transmembrane protein kinase activity, and other functions. Further KEGG analysis results showed that in addition to inflammation-related pathways, these differentially expressed genes were enriched in Parkinson's disease pathways, suggesting that sclarea alcohol may have the effect of treating Parkinson's disease. The specific results still need to be verified by subsequent experiments.

[0066] Example 2 Effects of sclareol on BV2 cell activity and release of inflammatory factors

[0067] 1. Cell recovery

[0068] 1) Preheat the culture medium in a 37°C water bath, take the BV2 cells out of the liquid nitrogen, immediately put them in a clean sealed bag and thaw them in a water bath. Gently shake the cryovials during the thawing process, but be careful not to shake the water in the water bath into the sealed bag.

[0069] 2) When the liquid in the cryovial is completely thawed, add 5 times the volume of complete culture medium and gently pipette to mix the cells.

[0070] 3) Set the centrifuge parameters to 1000 rpm and centrifuge for 5 min. After centrifugation, resuspend the cells gently with complete culture medium and place them in an incubator for 24 hours before observation.

[0071] 4) Freshly revived BV2 cells can be used directly in experiments without waiting for two generations.

[0072] 2. Cell passaging: Use DMEM medium containing 10% FBS for culture (special medium for BV2 cells; Pronocell CM-0493), place in a cell culture incubator at 37°C and 5% CO2 saturated humidity, wait until the cell confluence reaches 70-90%, and pass at a ratio of 1:4.

[0073] 3. Cell grouping and determination of experimental concentration

[0074] After passaged BV2 cells (ATCC / Sciencell) adhered to the wall, the culture medium was replaced and the cells were divided into Control group, Scl group, LPS group and LPS+Scl group. LPS 1 μg / ml was used to co-culture with microglia to simulate the inflammatory environment. The experimental cells were divided into the Control group without any treatment, the Scl group with sclareol and DMSO, the LPS group with LPS and DMSO, and the LPS+Scl group with LPS and sclareol. After culturing for 6 / 24 hours, the therapeutic effect of sclareol was investigated.

[0075] CCK8 cell viability test: After BV2 cells were added with LPS and cultured for 24 hours, the original culture medium was discarded, PBS was added and gently washed twice, 100 μL of 1% CCK8 solution was added to each well, cultured at 37°C for 30 minutes, and the absorbance was measured at 450 nm. Cell viability (%) = [A (drug added) - A (blank)] / [A (0 drug added) - A (blank)] * 100. The kit used was CCK8 detection kit (Beijing Solebao Company).

[0076] NO concentration measurement: After BV2 cells were cultured with LPS for 24 hours, the supernatant was aspirated, and NO detection reagent was added. The absorbance was measured at 540 nm and compared with the NO standard curve. The kit used was the NO detection kit (Shanghai Bioscan Biotechnology Co., Ltd.).

[0077] Depend on Figure 6It was shown that sclareol did not inhibit the viability of BV2 cells at a concentration of less than 60 μM. After the addition of LPS, the cell viability of microglia increased, which may be due to the fact that LPS stimulated the division and proliferation of BV2 cells. At the same time, the effect of the co-existence of LPS and sclareol on the viability of BV2 cells was determined. According to the experimental results, under the condition of the co-presence of LPS and sclareol, sclareol did not inhibit the viability of BV2 cells at a concentration of less than 60 μM. After LPS and sclareol were co-cultured with microglia for 24 hours, the NO concentration in the supernatant was detected using a NO detection kit. The action concentration of sclareol was selected to be 10-60 μM. According to the experimental results, sclareol has a better anti-inflammatory effect at a concentration of 40 μM. Therefore, the subsequent experimental study adopted a concentration of 40 μM.

[0078] 4. Detection of inflammatory factor levels

[0079] (1) The qPCR experiment was as described in Example 1.

[0080] (2) Immunoblotting

[0081] 1) Tissue and cell sampling

[0082] Tissue sampling: After all behavioral experiments in each group were completed, the mice were anesthetized by overdose for tissue sampling. The substantia nigra pars compacta and striatum of each group of mice were sampled on ice using a brain mold, and the tissues were placed in EP tubes, and the mouse numbers and brain tissues were marked.

[0083] Cell collection: gently blow the cells to collect the cells, centrifuge, discard the culture medium, add DPBS to resuspend, centrifuge again, and collect the cell pellet.

[0084] 2) Whole protein extraction

[0085] Total protein extraction from tissue: a) Add 100x protease inhibitors and acidase inhibitors to the enhanced RIPA lysis buffer according to the proportion, and place on ice for later use. b) Add 10μL RIPA lysis mixed solution for every 1mg of tissue, ultrasonically break the homogenate on ice to fully lyse, and let it stand for 30min. c) Centrifuge at 12,000×g for 20min at 4℃, take the protein supernatant and add it to a new centrifuge tube. d) Determine the protein concentration using the BCA quantitative method. e) Use RIPA lysis buffer and 5× loading buffer to adjust the protein concentration to 20μg / μl. After denaturation in a 95℃ water bath for 10min, the protein sample is then divided and stored at -80℃ for later use.

[0086] Extraction of whole cell proteins: a) Add 100x protease inhibitors and phosphatase inhibitors to enhanced RIPA lysis buffer according to the proportion and place on ice for later use. b) For every 1.0×10 6Add 50μl RIPA lysis mixed solution to each cell, ultrasonically break the homogenate on ice to fully lyse, and let it stand for 30min. c) Centrifuge at 12,000×g for 20min at 4℃, take the supernatant and add it to a new EP tube. d) Determine the protein concentration using the BCA quantitative method. e) Use RIPA lysis buffer and 5× loading buffer to balance the protein concentration to 20μg / μl. After denaturation in a 95℃ water bath for 10min, divide the protein sample and store it at -80℃ for later use.

[0087] BCA quantification: a) After diluting the protein sample to an appropriate concentration, add 20μl to each well of the 96-well plate sample well, and add 20μl BSA standard to the standard curve wells in the order of 0, 125, 250, 500, 750, 1000, 1500, and 2000. b) Mix the A and B solutions of the protein quantification kit in a ratio of 50:1 to form a protein quantification working solution, and add 200μl of protein quantification working solution to each well. c) Incubate at 37℃ for 30min. d) After incubation, detect the absorbance of the sample at 562nm. According to the corresponding relationship between the standard and the absorbance, prepare a standard curve to obtain the sample concentration.

[0088] SDS-PAGE gel electrophoresis: a) Add the protein sample and protein marker to the lane of the pre-prepared 15% SDS-PAGE gel (15% SDS-PAGE gel rapid preparation kit, Beijing Bio-Tech Biotechnology Co., Ltd.). b) First adjust the voltage to 70V, and when the bromophenol blue band reaches the boundary between the stacking gel and the separation gel and the protein marker band is clearly separated, adjust the voltage to 110V, and stop the electrophoresis when the marker near the target protein is completely separated.

[0089] Transfer: a) Prepare 1× electrotransfer buffer in advance and precool it in a 4°C refrigerator. b) Take out the gel and place it in the electrotransfer buffer, and cut off the excess part according to the marker. c) Place the sponge pad, filter paper, SDS-PAGE gel, NC membrane, filter paper, and sponge pad in the order from negative to positive, install the transfer device, use a roller to remove bubbles, and transfer the membrane at a constant current of 250mA in an ice bath for 45 minutes.

[0090] Antibody incubation: a) Immerse the NC membrane in 5% skim milk powder and block it on a shaker at room temperature for about 1 hour. b) Discard the skim milk, place the NC membrane in the primary antibody diluent, and incubate it on a shaker at 4°C overnight. c) Place the NC membrane in 1×TBST, shake at room temperature, and wash it for 5 minutes × 3 times. d) Place the washed NC membrane in the corresponding HRP secondary antibody diluent and incubate it on a shaker at room temperature for about 2 hours. e) Place the NC membrane in 1×TBST, shake at room temperature, and wash it for 5 minutes × 3 times.

[0091] ECL chemiluminescence: a) Mix solution A and solution B in the ECL luminescent solution in equal proportions. Immerse the NC membrane in the premixed ECL luminescent solution. b) Take out the NC membrane, drain the luminescent solution, and expose and photograph it in a luminometer. c) Quantify the grayscale value using ImageJ.

[0092] Depend on Figure 7 It was shown that sclareol reduced the level of inflammatory factors released by BV2 induced by LPS stimulation.

[0093] 5. Effect of sclareol on LPS-induced oxidative stress in BV2 cells

[0094] JC-1 mitochondrial membrane potential detection: Add CCCP diluted 1:1000 to the positive control group 24h in advance. Discard the culture medium, add the prepared JC-1 working solution, and incubate at 37℃ for 20min. Discard the working solution, wash twice with 1XJC1 buffer, then add 500μl culture medium, use confocal microscope to take images or detect by dual laser flow cytometer. The excitation light of JC-1 monomer is set to 488nm, and the emission wavelength is collected at 530±15nm. The excitation light of JC-1 aggregate is set to 550nm, and the emission wavelength is collected at 590±15nm. The kit used is JC-1 detection kit (Shanghai Beyotime Company).

[0095] Detection of intracellular ROS levels: A 1:2000 diluted Rosup was added to the positive control group in advance. After culturing for 30 minutes, the cells were collected, centrifuged, and the culture medium was discarded. The prepared H2DCFH-DA working solution was added and incubated at 37°C for 30 minutes, shaking every 3-5 minutes to ensure that the probe and the cells were in full contact. Then centrifuged, resuspended and centrifuged and washed 3 times with serum-free culture medium to fully remove the probe that did not enter the cells. Finally, the cells were resuspended in serum-free culture medium and detected using a dual-laser flow cytometer. The excitation light of the H2DCFH-DA probe was set to 488nm, and the emission wavelength was collected at 530±15nm. The kit used was the ROS detection kit (Shanghai Beyotime Company).

[0096] Intracellular glutathione peroxidase activity detection: collect cells by blowing, discard the culture medium after centrifugation, resuspend with PBS and centrifuge, discard PBS, and retain the cell pellet. Use RIPA lysis buffer to lyse the cells, centrifuge at 4°C, 12000rpm for 10min, and collect the supernatant into another clean EP tube. Prepare GPx detection working solution, add 40μl working solution to each well, and add 50μl sample to a 96-well plate. Immediately after adding 10μl of the prepared peroxide solution, use an enzyme reader to detect the absorbance at 340nm, and record it as the 0-minute reading. Set the temperature to 25°C, detect once every 1min, and record continuously for at least 5 minutes to obtain data at 6 points, and control the measured ΔA340 / min within the range of 0.01-0.2. According to [glutathione peroxidase activity in sample] = [glutathione peroxidase activity in detection system] × [dilution factor] / [protein concentration in sample] = [(ΔA340 / min) / (εμM × L (cm))] × [dil × (V (ml) / Vsample (ml))] / [protein concentration in sample], calculate the glutathione peroxidase activity in the sample. The kit used is GPx detection kit (Shanghai Beyotime Company).

[0097] like Figure 8 As shown, sclareol reduced the level of ROS in BV2 cells after LPS stimulation. Increased oxidative stress can be manifested as a decrease in mitochondrial membrane potential. JC-1 dye can selectively enter mitochondria and reversibly change from red to green as the membrane potential decreases. Sclareol inhibited the decrease in mitochondrial membrane potential in BV2 cells after LPS stimulation. Glutathione peroxidase (GPx) is an important antioxidant enzyme that is mainly responsible for clearing intracellular peroxides. Sclareol increased the activity of glutathione peroxidase in BV2 cells after LPS stimulation. The above experimental results suggest that sclareol has the effect of improving oxidative stress.

[0098] Example 3 Effect of sclareol on Parkinson's disease mice with overexpression of α-Syn in substantia nigra

[0099] 1. Animal modeling and grouping

[0100] The mouse was anesthetized with isoflurane gas and fixed on a stereotaxic brain injection instrument. The scalp near the injection site was prepared. After the skin preparation was completed, the skull was exposed using sterile surgical instruments. The position of the mouse head was adjusted so that the anterior and posterior fontanelles and the same sites on the left and right sides of the skull were kept level. The substantia nigra (AP: -3.0mm, ML: ±1.25mm, DV; -4.3mm from bregma) was determined, and a high-speed skull drill was used to drill through the skull at the needle site without damaging the pia mater. The needle was slowly inserted, and after reaching the target position, it was withdrawn 0.05mm, and the injection was set at a speed of 50nl / min. A total of 1μl of virus solution was injected, and the control virus was injected on the control side.

[0101] The mice were divided into Ctrl, Veh (ASO) and Scl (ASO) groups. 1 μl of HBAAV2 / 9-TH-h-SNCA-3xflag-Zsgreen (Hanheng Biotech (Shanghai) Co., Ltd.) was injected into the right substantia nigra, and the control virus HBAAV2 / 9-TH-ZsGreen (Hanheng Biotech (Shanghai) Co., Ltd.) was injected into the left substantia nigra. After 8 weeks, the virus was stably expressed. The Scl (ASO) group was given a quantitative diet of sclareol (Jiangsu Collaborative Bioengineering Co., Ltd.) 50 mg / Kg / day, and the mice were allowed to orally ingest it. Two weeks later, behavioral tests were performed and samples were collected.

[0102] 2. Behavioral testing

[0103] (1) Cylinder test: The mice were confined individually in a transparent plastic cylinder with a diameter of 20 cm and a height of 40 cm. After the mice entered the plastic cylinder, a video camera was used to record the number of times each mouse touched the cylinder wall within 3 minutes. The number of touches by the left limb alone, the right limb alone, and the number of touches by both limbs were recorded and counted. The frequency of use of the healthy forelimb of each mouse was then calculated.

[0104] (2) The pole climbing experiment is as described in Example 1.

[0105] (3) Rotarod test: Place the mouse on the rotarod and turn on the lowest speed of 4 rpm to allow the mouse to adapt. After adaptation, the rotarod begins to accelerate, evenly accelerating from 4 rpm to 40 rpm within 300 seconds, and start timing. Record the time before the mouse finally falls off the rotarod. Mice need to be trained continuously for two days before the formal experiment. Each mouse is tested three times and the median is taken.

[0106] The results are as follows Fig. 9As shown. The cylinder test showed that the virus-injected mice had an increased preference for the use of unilateral forelimbs, and this state was improved in mice that were orally treated with sclareol. The results of the pole climbing test showed that compared with the Veh (ASO) group, the mice had a shorter T turn time and a higher pole climbing speed. This suggests that the mice's motor and coordination abilities were improved. The results of the rotating rod test showed that the mice treated with sclareol had improved motor endurance and coordination.

[0107] 3. Immunostaining: The experimental steps are the same as those in Example 1.

[0108] After binarization of the microglial staining images in the substantia nigra, skeletonization analysis was performed. The number and length of branches were counted. The results are shown in Figure 2. Fig.10 In mice treated with sclareol, the number of branches of substantia nigra microglia increased and the length of branches prolonged compared with the Veh(ASO) group, suggesting that sclareol inhibited the activation of microglia.

[0109] After staining the TH+ neurons in the substantia nigra compacta of the modeled mice, the number of TH+ neurons was counted. Fig.11 The results showed that overexpression of α-Syn caused a decrease in the number of TH+ neurons, while the number of TH+ neurons in the substantia nigra of mice treated with sclareol was protected, suggesting that sclareol may have a protective effect on neurons. Combined with existing experimental results, it is speculated that this protective effect is most likely achieved by inhibiting microglia activation.

[0110] 4. Immunoblotting experiment: The experimental steps are the same as those in Example 2.

[0111] The substantia nigra of the modeling side of the mice was taken for immunoblotting experiments to detect the content of α-Syn and the level of inflammatory factors. Fig.12 The results showed that compared with the Veh(ASO) group, sclareol reduced the content of α-Syn in the substantia nigra. α-Syn overexpression caused an increase in local inflammatory factors, which was inhibited after oral administration of sclareol.

[0112] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of sclareol in the preparation of a drug for inhibiting neuroinflammation, characterized in that: The sclareol can improve the hypoactivity, memory loss and motor impairment caused by neuroinflammation in mice.

2. The use according to claim 1, characterized in that The sclareol reduced the increase of inflammatory factors in the mouse cortex and downregulated inflammation-related pathways.

3. The use according to claim 1, characterized in that The sclareol inhibits the activation of mouse cortical microglia.

4. The use according to claim 1, characterized in that The sclareol reduces the level of inflammatory factors released by BV2 cells caused by LPS stimulation and inhibits the level of oxidative stress of BV2 cells caused by LPS stimulation.

Images