Application of small molecule compound in relieving neuroinflammation of MLD disease

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of a small molecule compound in relieving neuroinflammation caused by MLD diseases. In order to develop a new treatment choice for MLD, an in-vitro mouse-derived OCS model is adopted for research, and it is found that Deltarasin can inhibit microglial cell hyperplasia and promote the microglial cell hyperplasia to present an amebic form, and meanwhile expression of neuroinflammatory factors such as proinflammatory cytokines and chemotactic factors is relieved. And various adverse effects caused by sulfate can be weakened by Deltarasin, which indicates that the Deltarasin has a nerve protection effect. Therefore, the Deltarasin, which is a small-molecule inhibitor, shows an important treatment value in the field of MLD diseases, and is worthy of further clinical research.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to application of a small molecule compound in reducing neuroinflammation of MLD disease. BACKGROUND

[0002] Metachromatic leukodystrophy (MLD) is a lysosomal disease caused by recessive mutations in the arylsulfatase A gene (ARSA), which encodes the arylsulfatase A enzyme (ASA). ASA is essential for the metabolism of galactosylceramide-3-O-sulfate (Sulfatide), and its deficiency leads to accumulation of Sulfatide in the central and peripheral nervous system, forming lysosomal storage deposits. Sulfatide is the most abundant sphingolipid in myelin, accounting for 4% of its composition, and plays an important role in maintaining myelin function. In MLD, Sulfatide substances accumulate in oligodendrocytes, Schwann cells, phagocytes, astrocytes, microglia and neurons, and further cause demyelination. This accumulation of Sulfatide substances is considered a key factor in neuroinflammation in MLD. MLD is associated with muscle loss, decreased cognitive function, and gradual loss of vision, with an estimated birth rate of about 1-2 / 100,000 and an incidence of about 1 / 40,000. This devastating demyelinating disease can be divided into late infantile, juvenile and adult types according to the age of onset. All types of MLD are characterized by a variety of neurological symptoms, which ultimately lead to death if left untreated. There is currently no cure for MLD, so there is an urgent need for new treatment options.

[0003] Sulfatide levels in cerebrospinal fluid and the peroneal nerve are associated with the severity of neuropathy in MLD patients, and studies have shown that elevated Sulfatide levels in cerebrospinal fluid are associated with worsening motor function in MLD patients. However, the mechanism by which Sulfatide induces demyelination in MLD is not yet clear. Increasing evidence suggests that the extensive demyelination and microglial loss observed in MLD may be due to chronic inflammation induced by Sulfatide, which is based on the excessive release of cytokines and chemokines.

[0004] Sulfatide is an important but relatively less studied immunomodulatory component in the central nervous system. The levels of pro-inflammatory factors are elevated in MLD patients, and the cytokines and chemokines in their cerebrospinal fluid include CCL2, IL-1Ra, IL-8 and CCL4. In addition, Sulfatide is known to have effects on a variety of immune cells, such as neutrophils, dendritic cells, B cells, and microglia in the central nervous system. However, it is not yet clear whether neuroinflammation is associated with MLD.

[0005] Deltarasin is a small molecule inhibitor that can inhibit the interaction of KRAS and PDEdelta, and its dissociation constant (K d ) for binding to pure PDEdelta is 38nM. Notably, this small molecule inhibitor has shown some efficacy in reducing inflammation in neurological diseases. Therefore, it is necessary to further develop the therapeutic potential of Deltarasin in metachromatic leukodystrophy (MLD). SUMMARY

[0006] In order to overcome the shortcomings of the prior art described above, in view of the fact that Deltarasin, a small molecule inhibitor, can inhibit the interaction of KRAS and PDEdelta, the present application studies the effect of Deltarasin on organotypic cerebellar slice culture models (OCS models) treated with sulfate, and finds that it can prevent neuroinflammation in MLD models through immunomodulation and direct neuroprotection, and is expected to be applied in the field of treating MLD diseases.

[0007] In order to achieve the above-mentioned purpose, the technical solution adopted by the present application is:

[0008] The present application provides the use of Deltarasin in the preparation of a medicament for treating metachromatic leukodystrophy (MLD).

[0009] The present application also provides the use of Deltarasin in the preparation of a medicament for treating demyelination in metachromatic leukodystrophy (MLD).

[0010] The present application has found that Deltarasin can prevent neuroinflammation in MLD models through immunomodulation and direct neuroprotection, and is expected to be applied in the treatment of MLD and neuroinflammation-related diseases.

[0011] Preferably, the Deltarasin exerts a therapeutic effect by reducing neuroinflammation in metachromatic leukodystrophy.

[0012] More preferably, the Deltarasin reduces neuroinflammation in metachromatic leukodystrophy by reducing the secretion of pro-inflammatory cytokines and chemokines.

[0013] Preferably, the effective concentration of the Deltarasin is 80-500nM.

[0014] Preferably, the medicament further comprises a pharmaceutically acceptable excipient.

[0015] More preferably, the adjuvants include at least one of excipients, propellants, solubilizers, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adherents, integrating agents, penetration promoters, pH regulators, buffers, plasticizers, surfactants, foaming agents, antifoaming agents, thickening agents, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculants, antioxidants, adsorbents, filter aids, release retarders.

[0016] More preferably, the dosage form of the drug includes tablets, capsules, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents or suppositories.

[0017] Compared with the prior art, the present application has the following beneficial effects:

[0018] MLD is a severe demyelinating disease, which belongs to autosomal recessive leukodystrophy. The disease is caused by ARSA gene mutation, which causes insufficient activity of ARSA lysosomal enzyme, and further causes accumulation of Sulfatide in the brain.

[0019] The present application develops research by using an in vitro mouse-derived OCS model, and finds that Deltarasin can inhibit microglial cell proliferation and promote it to present amoeba-like morphology, and at the same time, reduce the expression of neuroinflammatory factors such as pro-inflammatory cytokines and chemotactic factors. And various adverse effects caused by sulfatides can be weakened by Deltarasin, which shows that it has a neuroprotective effect.

[0020] In summary, the small molecule inhibitor Deltarasin shows important therapeutic value in the field of MLD disease, and is worthy of further clinical research. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1Deltarasin attenuates Sulfatide-induced microglial response; A: immunofluorescence staining images showing Ibal (yellow, labeling microglia) and Hoechst (blue, labeling nuclei) immunostaining results of microglia under different treatment groups; B: bar graph showing the change of Ibal fluorescence intensity after sulfatide (20 mM) alone and combined with Deltarasin (100 nM) treatment for 24 hours; data is expressed as a percentage of the control group; C: bar chart with the vertical coordinate being the number of cells (per mm2) for counting the number of microglia in different treatment groups, Ibal positive cell counting shows that sulfatide promotes microglial proliferation, and Deltarasin inhibits this effect; D: representative confocal images showing Ibal immunostaining results; E: cell surface Ibal quantity quantitative analysis shows that the expression of Ibal in sulfatide treatment group is significantly increased, but this effect is attenuated after Deltarasin treatment; F-G: Western blotting was used to detect the change of Ibal expression level, and the bar graph shows that the protein level of Ibal increases after sulfatide exposure, and this effect is attenuated after Deltarasin treatment.

[0022] Figure 2 Sulfatide increases cytokine and chemokine release in OCS model, after 24 hours, the release of cytokines and chemokines in the OCS model of the control group and the Sulfatide stimulated group was measured; among them, Sulfatide (10 mM, 20 mM, 50 mM and 100 mM) significantly increased the release of (A) IL-6, (B) TNF-a, (C) IL-17A, (D) CCL3, (E) MIF and (F) IFN-g, and these data were determined by ELISA.

[0023] Figure 3 Deltarasin regulates the release of cytokines and chemokines in Sulfatide treated OCS model, the release of cytokines and chemokines was measured after the control group and Sulfatide stimulated (10 mM, 20 mM) OCS model (transparent bar) was exposed to Deltarasin for 24 hours (black bar); Deltarasin (100 nM) significantly reduced the release of (A) IL-6, (B) TNF-a, (C) IL-17A, (D) CCL3, (E) MIF and (F) IFN-g, and these data were determined by ELISA. DETAILED DESCRIPTION

[0024] The specific embodiments of the present application are further described below. It is to be understood that the description of these embodiments is intended for purposes of illustration only and is not intended to be limiting. Furthermore, the technology features involved in the various embodiments of the present application described below can be combined in any combination provided that the combinations do not result in contradictions.

[0025] The experimental methods in the following examples are all conventional methods unless otherwise specified. The experimental materials used in the following examples are all commercially available unless otherwise specified.

[0026] The present application is studied by using an in vitro mouse-derived OCS model. The research data show that the OCS model treated with Sulfatide presents an enhanced performance of inflammatory characteristics, which is embodied in the enhanced proliferation / activation of microglia cells, and the increased levels of pro-inflammatory cytokines and chemokines such as IL-6, IL-17A, TNF-α, CCL3, MIF, IFN-γ. The activation of pro-inflammatory pathways in the brain (including IL-6 pathway, TNF-α, IL-17A and MIF) is the potential intersection between chronic neuroinflammation and various brain diseases such as Alzheimer's disease and multiple sclerosis. Therefore, it is reasonably speculated that the increase of these cytokines helps the demyelination process in MLD. In addition, the observed increase in CCL3, IFN-γ and IL-6 levels in the present application is consistent with the increase in the preclinical stage of MLD. It is generally believed that these cytokines promote pathological processes by activating microglia cells, recruiting peripheral immune cells such as granulocytes and T cells. CCL3, IFN-γ and TNF-α, as type 1 helper T cell cytokines, also play a pathogenic role in other demyelinating diseases such as multiple sclerosis. They can drive the recruitment and activation of immune cells and have toxic or pro-apoptotic effects on microglia cells. Studies have shown that the production of IL-17A is a key factor in autoimmune demyelination, and is almost completely induced by high levels of IL-6 helper T cell 17 cytokines. Therefore, the present application further supports the view that neuroinflammation may be a major driving factor in the pathogenesis of MLD, and by regulating immune pathways and microglia cells, it is expected to improve the level of myelination and reduce axonal degeneration.

[0027] Further research revealed that sulfatide induced demyelination in an OCS model in a concentration-dependent manner, and that sulfatide-treated OCS models also impaired microglial survival and axonal morphology. Deltarasin, however, demonstrated potent efficacy in rescuing sulfate-induced demyelination and axonal degeneration caused by sulfate treatment. Furthermore, sulfate increases the secretion of pro-inflammatory cytokines and chemokines such as IL-6, IL-17A, TNF-α, CCL3, MIF, and IFN-γ, a phenomenon that could be inhibited by deltarasin. Finally, data showed that deltarasin attenuated the enhanced microglial proliferation / activation and damage observed in sulfatide-treated OCS models.

[0028] It remains unclear whether deltarasin exerts its beneficial effects directly on oligodendrocytes or indirectly by protecting myelinated axons; in fact, both are possible. Importantly, deltarasin significantly reduced the inflammatory response associated with demyelination, specifically by decreased proliferation and activation of Iba1 in microglia and reduced levels of pro-inflammatory molecules. These studies collectively demonstrate that deltarasin prevents neuroinflammation in MLD models through immunomodulation and direct neuroprotection, providing strong evidence for its application in MLD and neuroinflammation-related diseases. Further understanding of deltarasin's function in brain homeostasis and its dysregulation under pathological conditions may contribute to the development of clinical treatments for MLD.

[0029] To fully and clearly present the technical solution and significant advantages of the present invention, the present invention will be described in detail below with reference to specific embodiments.

[0030] 1. Experimental Methods

[0031] 1.1 Pharmacological compounds

[0032] Sulfatide is the main glycolipid component of myelin, prepared as a 10 mM stock solution dissolved in DMSO. Deltarasin is a small molecule inhibitor that inhibits the interaction between KRAS and PDEδ: it binds to pure PDEδ K... d The value is 38 nM; in hepatocytes, K inhibits the interaction between RAS and PDEδ. dThe value is 41 nM. By inhibiting the interaction of PDE5 with KRAS, Deltarasin can inhibit the proliferation of human pancreatic ductal adenocarcinoma cells dependent on oncogenic KRAS. In a nude mouse xenograft model inoculated subcutaneously with human Panc-Tu-I tumor cells, Deltarasin (10 mg / kg, intraperitoneal injection) can dose-dependently inhibit tumor growth.

[0033] The stock solution of Deltarasin is prepared by dissolving 1 mg of Deltarasin in 1.6563 mL of DMSO to make a 1 mM stock solution. The stock solution should be stored in a -80°C refrigerator, and should be stored in separate containers to avoid repeated freezing and thawing. When used, dilute to the working solution concentration.

[0034] 1.2, Establishment of sulfatide-induced demyelination OCS model

[0035] The mouse organotypic cerebellar slice (OCS) culture model is prepared from postnatal day 10 (P10) C57BL / 6 mice provided by the Second People's Hospital of Shenzhen. All tissue isolation procedures follow the protocols approved by the Animal Ethics Committee of the Second People's Hospital of Shenzhen.

[0036] The OCS model experiment process is as follows: decapitate the mouse, separate the cerebellum from the hindbrain after removing the skull; use a McIlwan tissue slicer to cut the cerebellum into 400 μm thick sagittal sections; place the tissue in Opti-MEM and separate it into individual sections under a dissecting microscope; place 5 sections in each cell culture insert and culture at 35.5°C, 95% humidity and 5% CO2.

[0037] The use of culture medium needs to be adjusted according to the stage: for the first 4 days of culture, use a culture medium containing 50% Opti-MEM, 25% Hank's Balanced Salt Solution (HBSS) and 25% heat-inactivated horse serum, which needs to be supplemented with 2 mM Glutamax, 28 mM D-glucose, 1% penicillin / streptomycin and HEPES; replace with serum-free medium on the 4th day, which contains 96% Neurobasal A, 2% B-27 supplement, supplemented with 1% penicillin / streptomycin, 28 mM D-glucose, 2 mM Glutamax and 10 mM HEPES; replace with the above serum-free medium again on the 10th day.

[0038] To induce demyelination in the OCS model, treat the sections with 20 μM sulfatide for 24 hours on the 12th day (at this concentration, the process of demyelination can be rescued as neuronal degeneration has not yet occurred).

[0039] 1.3, Rescue effect of Deltarasin in sulfatide-induced demyelination mouse OCS model

[0040] To investigate the therapeutic effect of Deltarasin in Sulfatide-induced demyelination disease, on day 12 of the above mouse organotypic cerebellar slice (OCS) model, the following treatment was performed: the OCS model was incubated with Sulfatide at different concentrations in the presence or absence of 100 nm Deltarasin, at 37°C in a 5% CO2 incubator for 24 hours. The specific grouping is as follows:

[0041] Control group (control): untreated OCS model (including untreated Sulfatide + Deltarasin group and only Deltarasin treated group); experimental group: OCS model treated with Sulfatide at different concentrations in the presence or absence of 100 nm Deltarasin.

[0042] By comparing the differences in demyelination-related indicators between the two groups of slices, the therapeutic effect of Deltarasin was evaluated.

[0043] 1.4, Immunofluorescence of OCS model

[0044] After the end of Sulfatide / Deltarasin treatment, OCS models were fixed with 4% paraformaldehyde (PFA) for 7 minutes, followed by two washes with phosphate-buffered saline (PBS) for 10 minutes each; blocking and permeabilization were performed overnight at 4°C in PBS containing 10% bovine serum albumin (BSA) + 0.5% Triton X-100; primary antibodies were diluted in PBS containing 2% BSA + 0.1% Triton X-100 and incubated for 48 hours at 4°C (primary antibodies included rabbit anti-myelin basic protein (MBP), mouse monoclonal anti-myelin oligodendrocyte glycoprotein (MOG), chicken anti-neurofilament heavy (NFH), mouse monoclonal anti-vimentin, rabbit anti-ionized calcium-binding adapter molecule 1 (Iba1), chicken anti-glial fibrillary acidic protein (GFAP), etc.); after this, sections were washed with PBS for 5 minutes and then with PBS + 0.1% Triton X-100 buffer for 10 minutes three times; secondary antibodies were incubated for 18 hours at 4°C (secondary antibodies used included goat anti-rabbit Alexa Fluor 488, goat anti-mouse Alexa fluor 488, and goat anti-chicken IgY Alexa Fluor 633); after re-washing the sections, they were stained with Hoechst nuclear stain (diluted 1 : 10,000 in PBS) and mounted with Gold anti-quenching reagent; samples were kept in the dark at 4°C until imaging, and images were captured by confocal microscopy at 1024 x 1024 resolution, at a speed of 200 frames per second (fps). Gold anti-quenching reagent; samples were kept in the dark at 4°C until imaging, and images were captured by confocal microscopy at 1024 x 1024 resolution, at a speed of 200 frames per second (fps).

[0045] 1.5. Microscopy and image analysis of OCS models

[0046] Immunofluorescence images of OCS models were taken at x20 or x40 magnification using a Leica SP8 confocal microscope. Each experimental group (n=5) contained 5 sections, and 5-6 fluorescent images were taken per section; the cerebellar regions captured for each treatment group were consistent and covered a large portion of the total cerebellar area. Images were exported as 8-bit.tif files for analysis using the FIJI software package. Intensity values were normalized to the mean of the control group for each experiment and each marker.

[0047] Analysis of SMI-32 expression in white matter tracts: Regions of interest (ROI) containing mainly white matter tracts were manually selected in each image, the fluorescence intensity was calculated, and the proportion of SMI-32 immunoreactive parts (referred to as SMI-32 surface area) in the white matter region was quantified.

[0048] Fluorescence image processing for PARP-1: The ROI of the cell nucleus in the Hoechst-stained field was automatically generated by ImageJ, and the average gray value of the region was quantified.

[0049] Cell counting and surface area analysis using the particle analysis tool of FIJI: Convert the 8-bit image to a binary image, set 20 pixels as the minimum particle size; generate a detected particle mask to verify the accuracy of the detection method, and then analyze the numbered list showing the fluorescence intensity and the area of each particle.

[0050] Analysis of astrocyte morphology: Use the skeletonization tool of FIJI and the AnalyzeSkeleton (2D / 3D) software plug-in to summarize the cell morphology by the number of astrocyte branches output by the plug-in.

[0051] 1.6, blotting hybridization test

[0052] After sulfatide / deltarasin treatment, the OCS model was homogenized in RIPA buffer containing protease inhibitors (cOmplete) and sonicated, then centrifuged at 14,000 rpm, and the supernatant was collected. After denaturation, electrophoresis was performed on a 12% SDS-polyacrylamide gel; after electrophoresis, the proteins were transferred to a PVDF membrane by semi-dry transfer method, and blocked with 5% BSA in PBS+0.05% Tween buffer for 1 hour at room temperature. The primary antibody was incubated overnight at 4°C, and the primary antibody used included rabbit anti-MBP, mouse monoclonal anti-MOG, mouse monoclonal anti-vimentin, rabbit anti-Iba1, chicken anti-GFAP and rabbit anti-Olig2. After washing the membrane, the HRP-labeled secondary antibody (goat anti-rabbit, donkey anti-chicken or goat anti-mouse) was incubated at room temperature for 2 hours. Then the membrane was developed using a chemiluminescent HRP substrate, and the image was acquired by C-Digit blot scanner and Image Studio4.0 (LI-COR).

[0053] 1.7, enzyme-linked immunosorbent assay (ELISA) quantification of inflammatory secretions

[0054] OCS models were treated with Sulfatide drugs (in the presence or absence of Deltarasin) for 24 hours, after which the culture medium was collected and stored at -80°C. The levels of interleukin 6 (IL-6), tumor necrosis factor-a (TNF-a), interleukin 17A (IL-17A), macrophage inflammatory protein-1a (MIP-1a / CCL3), macrophage migration inhibitory factor (MIF), and interferon-g (IFN-g) in the conditioned medium were then detected using corresponding mouse ELISA kits: IL-6 ELISA kit (R&D Systems; DY406), mouse TNF-a kit (R&D Systems; DY410), mouse IL-17A kit (ELISA Genie; MOFI01286), mouse CCL3 kit (R&D Systems; DY450), mouse MIF kit (R&D Systems; DY1978), and mouse IFN-g kit (ELISA Genie; MOFI00047), respectively, and were operated in strict accordance with the manufacturer's instructions.

[0055] The quantitative analysis of IL-6, TNF-a, CCL3, and MIF secreted in the conditioned medium was performed according to the R&D Systems kit instructions, with the specific steps as follows:

[0056] (1) Coat a 96-well ELISA plate with capture antibody diluted in PBS, and incubate at 4°C overnight; (2) discard the liquid in the wells, wash the plate three times with washing buffer (10-fold PBS containing 0.05% Tween 20, pH 7.4), and then block with appropriate reagent diluent at room temperature for 2 hours; (3) discard the blocking solution, wash the plate again three times with washing buffer, and aspirate the residual buffer in the wells; (4) prepare a standard curve by serial dilution of recombinant protein with appropriate reagent diluent; add samples and standards to the antibody-coated ELISA plate, and incubate at room temperature for 2 hours; (5) discard the liquid in the wells, wash the plate three times with washing buffer, and then add detection antibody diluted in reagent diluent to each well, and incubate at room temperature for 2 hours; (6) discard the detection antibody, wash the plate three times with washing buffer, and add diluted color developing agent (R&D systems; DY999) to each well, and incubate at room temperature in the dark for 20 minutes; (7) wash the plate again three times with washing buffer, add substrate solution to the wells, and incubate at room temperature in the dark for 20 minutes; (8) terminate the color developing reaction by adding 1M sulfuric acid, and immediately read the absorbance at 450nm using an enzyme reader (Labsystem Multiskan).

[0057] For IL-17A and IFN-γ detection, the operation was as follows: using pre-coated plates, washing three times before adding standards and samples, then adding standards and samples to the plates according to the manufacturer's instructions, incubating at room temperature for 2 hours; the standard curve was plotted by plotting the standard concentration against the corresponding absorbance value, and the cytokine level was calculated according to the standard curve, and the result was expressed in pg / mL.

[0058] 1.8. Statistical analysis

[0059] All data were analyzed using the GraphPad Prism 5 software package (GraphPad Software, Inc.). The normality of the data was determined by the Shapiro-Wilk test. Where applicable, histological and biochemical data were analyzed using parametric one-way or two-way ANOVA with Tukey's post-hoc test for multiple comparisons to assess numerical differences between control, Sulfatide-treated, and Deltarasin-treated OCS models. For Western blot experiments, one-way ANOVA with Newman-Keuls post-hoc test was used to compare all groups to each other.

[0060] The mean fluorescence intensity measured using ImageJ software was expressed in arbitrary units. The raw data set was normalized and presented as a percentage of the control mean value. Differences were considered statistically significant if p < 0.05, and all values were expressed as "mean ± standard error" (SEM).

[0061] "n" represents the number of independent organotypic slice cultures prepared on different experimental days. Each experiment included 5 technical replicates for each treatment group, with experiment replicates n = 3, 4, or 5. Different experiments used slices from different brain tissues from different mouse litters. For each slice culture, 25 healthy slices were obtained from the cerebellum of 5 littermates and randomly assigned to 5 different treatment groups. Therefore, when the experiment was repeated n = 5 times, a total of 25 OCS models were stained and imaged.

[0062] 2. Experimental results

[0063] 2.1. Deltarasin reduces Sulfatide-induced microglial activation

[0064] The present invention studies the effect of Sulfatide and Deltarasin on microglial cells. Representative confocal images show Ibal (yellow) and Hoechst (blue) immunostaining under treatment conditions after OCS model was treated with 20 mM Sulfatide. There was a significant increase in microglial activation as reflected by the fluorescence intensity of ionized calcium binding adapter molecule 1 (Ibal) (data expressed as percentage of control group) (451.9% vs. 100% for control group; *** p<0.001, compared to control group) Figure 1 A and B). More importantly, these changes in Ibal fluorescence were reduced by Deltarasin (100 nM) treatment (Sulfatide: 451.9% vs. Sulfatide + Deltarasin: 110.1%, *** p<0.001 Figure 1 B). Furthermore, Deltarasin also reduced microglial proliferation induced by Sulfatide as reflected by Ibal cell count, which showed that Sulfatide can promote microglial proliferation, while Deltarasin can reduce microglial proliferation (Sulfatide: 32.3 vs. Sulfatide + Deltarasin: 18.5, *** p<0.001 Figure 1 C). Given the clear association between microglial morphology and their function, i.e. when they have short and thin processes and appear branched they are considered "resting", while when they appear spherical and lack processes they are considered "activated", the effect of Sulfatide and Deltarasin on microglial morphology was investigated. Analysis of Ibal total surface area vs. cell number showed that when OCS model was treated with 20 mM Sulfatide, there was a significant increase in cell number (Control: 2.29 vs. Sulfatide: 3.38, * p<0.05). Deltarasin alone did not have a significant effect on Ibal volume, while when Sulfatide was used in combination with Deltarasin, it significantly reduced the area of microglial surface, which indicates that Deltarasin is able to attenuate Sulfatide-induced microglial activation (Sulfatide: 3.38 vs. Sulfatide + Deltarasin: 1.92, *** p<0.001 Figure 1(D and 1E). Furthermore, Deltrasin can attenuate the increase in total Iba1 levels caused by Sulfatide, which is also reflected in the Western blot, with data expressed as a percentage of the control group (Sulfatide: 410.9%). ** p<0.01, compared with the control; Sulfatide+Deltarasin: 129.5%, * p<0.05, compared with Sulfatide)( Figure 1 These results indicate that Iba1 expression and activation are increased in Sulfatide-treated OCS, while Deltrasin significantly attenuates this increase in Iba1.

[0065] 2.2 Sulfatide led to an increase in cytokines and chemokines in cerebellar slices.

[0066] MLD patients exhibit elevated levels of pro-inflammatory cytokines and chemokines, primarily including CCL3 (CC motif chemokine ligand 3), IL-1Ra (Interleukin-1Ra), IL-8 (Interleukin-8), and CCL4 in cerebrospinal fluid. Given that the above studies indicate that sulfatide can induce microglial cell activation, we then investigated its effects on the secretion of pro-inflammatory cytokines and chemokines in an OCS model. After treating the OCS model with Sulfatide (10 μM, 20 μM, 50 μM, 100 μM) for 24 hours, the levels of soluble IL-6 (Interleukin-6), IL-17A (Interleukin-17A), TNF-α (Tumor Necrosis Factor-α), CCL3, MIF (migration inhibitory factor), and IFN-γ (interferon-γ) were significantly increased. The expression of pro-inflammatory cytokines or chemokines at 10 μM and 20 μM Sulfatide doses compared to the untreated control group was as follows: IL-6 (Control: 8.2 pg / mL; 10 μM: 182.4 pg / mL, **p<0.01; 20 μM: 166.5 pg / mL, *p<0.05); Figure 2A), TNF-a (Control: 22.3 pg / mL; 10 mM: 110.4 pg / mL, ***p<0.001; 20 mM: 118.9 pg / mL, ***p<0.001; Figure 2 B), IL-17A (Control: 61.5 pg / mL; 10 mM: 769.4 pg / mL, ***p<0.001; 20 mM: 762.2 pg / mL, ***p<0.001; Figure 2 C), CCL3 (Control: 64.7 pg / mL; 10 mM: 572.2 pg / mL, *p<0.05; 20 mM: 629.3 pg / mL, **p<0.01; Figure 2 D), MIF (Control: 869.2 pg / mL; 10 mM: 2949.4 pg / mL, ***p<0.001; 20 mM: 2287.0 pg / mL, *p<0.05; Figure 2 E) and IFN-g (Control: 36.6 pg / mL; 10 mM: 711.8 pg / mL, ***p<0.001; 20 mM: 426.8 pg / mL, **p<0.01; Figure 2 F). These data indicate that exposure of OCS model to Sulfatide, in addition to causing increased demyelination and axonal damage, also promotes inflammatory response.

[0067] 2.3, Deltarasin significantly reduces Sulfatide-induced release of proinflammatory cytokines and chemokines in OCS model

[0068] Given the above studies showing that Deltarasin can attenuate Sulfatide-induced microglial activation, it was further investigated whether Deltarasin can inhibit Sulfatide-induced release of inflammatory cytokines and chemokines. Treatment of OCS model with 100 nM Deltarasin for 24 hours significantly reduced Sulfatide-induced release of IL-6 (Control: 8.8 pg / mL vs. Deltarasin: 4.4 pg / mL; 10 mM 182.4 pg / mL vs. 21.6 pg / mL, *p<0.05; 20 mM 166.5 pg / mL vs. 27.7 pg / mL, *p<0.05; Figure 3A), TNF-a (Control: 22.3 pg / mL vs. Deltarasin: 16.0 pg / mL; 10 mM 110.4 pg / mL vs. 59.1 pg / mL, *p<0.05; 20 mM 118.9 pg / mL vs. 70.2 pg / mL, *p<0.05; Figure 3 B), IL-17A (Control: 61.5 pg / mL vs. Deltarasin: 37.2 pg / mL; 10 mM 1769.4 pg / mL vs. 93.0 pg / mL, ***p<0.001; 20 mM: 762.2 pg / mL vs. 82.2 pg / mL, ***p<0.001; Figure 3 C), CCL3 (Control: 64.7 pg / mL vs. Deltarasin: 87.8 pg / mL; 10 mM: 575.2 pg / mL vs. 442.5 pg / mL, **p<0.01; 20 mM: 629.3 pg / mL vs. 423.2 pg / mL, **p<0.01; Figure 3 D), MIF (Control: 869.2 pg / mL vs. Deltarasin; 885.0 pg / mL; 10 mM: 2919.4 pg / mL vs. 1695.4 pg / mL, **p<0.01; 20 mM: 2317.0 pg / mL vs. 1259.2 pg / mL, *p<0.05; Figure 3 E), and IFN-g (Control: 36.6 pg / mL vs. Deltarasin: 57.6 pg / mL; 10 mM: 711.8 pg / mL vs. 81 pg / mL, ***p<0.001; 20 mM: 426.8 pg / mL vs. 49.3 pg / mL, ***p<0.001; Figure 3 F). These data show that sulfatide induction promotes neurogenic responses, while Deltarasin can attenuate this neuroinflammation by significantly reducing the secretion of proinflammatory cytokines and chemokines.

[0069] The embodiments of the present application have been described in detail, but the present application is not limited to the described embodiments. Various changes, modifications, substitutions and variations of these embodiments can be made by those skilled in the art without departing from the principles and spirit of the present application, and still fall within the scope of the present application.

Claims

1. Use of Deltarasin in the preparation of a medicament for treating metachromatic leukodystrophy.

2. Use of Deltarasin in the preparation of a medicament for treating demyelination of metachromatic leukodystrophy.

3. Use according to claim 1 or 2, characterized in that, The Deltarasin exerts a therapeutic effect by reducing neuroinflammation of metachromatic leukodystrophy.

4. Use according to claim 3, characterized in that, The Deltarasin reduces neuroinflammation of metachromatic leukodystrophy by reducing the secretion of proinflammatory cytokines and chemokines.

5. Use according to claim 1 or 2, characterized in that, The effective concentration of the Deltarasin is 80-500 nM.

6. Use according to claim 1 or 2, characterized in that, The medicament further comprises a pharmaceutically acceptable excipient.

7. Use according to claim 6, characterized in that, The excipient comprises at least one of an excipient, a propellant, a solubilizer, a cosolvent, an emulsifier, a colorant, a binder, a disintegrant, a filler, a lubricant, a wetting agent, an osmotic pressure regulator, a stabilizer, a glidant, a flavoring agent, a preservative, a suspending agent, a coating material, an aromatic agent, an antiadherent, an integrating agent, a penetration enhancer, a pH regulator, a buffer, a plasticizer, a surfactant, a foaming agent, an antifoaming agent, a thickening agent, a complexing agent, a humectant, an absorbent, a diluent, a flocculating and deflocculating agent, an antioxidant, an adsorbent, a filter aid, a release retardant.

8. Use according to claim 6, characterized in that, The dosage form of the medicament comprises a tablet, a capsule, an aerosol, a pill, a powder, a solution, a suspension, an emulsion, a granule, a liposome, a transdermal agent, or a suppository.

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