New application of tocopherol

Abstract

The present application relates to a new use of chloranil. The present invention provides an application of Impatiens balsamina extract or Lawsone in the preparation of a medicament for treating an autoimmune disease, and the present invention also provides a method for treating an autoimmune disease (especially MS or EAE). The use of Impatiens balsamina extract or Lawsone can effectively alleviate MS or EAE, reduce the proportion of Th1 cells, and do not affect immune homeostasis. At the same time, the present invention reveals the immune mechanism of the natural compound Lawsone in regulating MS, providing a new therapeutic target and theoretical basis for basic research and clinical treatment of MS.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to application of Impatiens balsamina extract or Lawsone in preparing medicines for treating autoimmune diseases. Background Art

[0002] The magical nature contains the richest chemical diversity. Most traditional Chinese medicines come from natural plants. The development of Chinese and foreign medical drugs has always been closely related to the development and research of natural small molecule compounds. Henna, also known as Impatiens balsamina, is the whole herb of Impatiens balsamina L., a plant of the Impatiens family. Impatiens balsamina mainly contains flavonols and naphthoquinone derivatives, such as 2-methoxy-1,4-naphthoquinone, kaempferol-3-rhamnosyl diglucoside, 2-hydroxy-1,4-naphthol, etc. The root of Impatiens balsamina contains lawsone, 2-methoxy-1,4-naphthoquinone, scopolamine, isofragin, spinasterol, etc., which have antifungal, antiallergic, anti-inflammatory, analgesic, and skin dialysis-promoting effects.

[0003] Multiple sclerosis (MS) is a typical autoimmune disease caused by T-cell-mediated immune cells, characterized by central nervous system (CNS) inflammation, demyelinating lesions and nerve damage. The main clinical symptoms are muscle weakness, limb sensory impairment, visual impairment, cognitive impairment, depression, dizziness, ataxia, bladder or rectal dysfunction, etc. A small number of patients have epilepsy and intellectual disabilities. It is the leading cause of non-traumatic paralysis in young and middle-aged people and is called "immortal cancer". Experimental autoimmune encephalomyelitis (EAE) is widely used as an ideal animal model for studying MS because its pathological characteristics are very similar to those of MS. In May 2018, MS was included in China's "First List of Rare Diseases". In August 2020, The Lancet Regional Health magazine reported the incidence of MS in my country, filling the gap in China's MS epidemiological data. Studies have shown that the incidence rate among children in my country is 0.055 / 100,000, and the incidence rate among adults is 0.288 / 100,000. The peak age of onset is 40-49 years old, which seriously affects the quality of life of patients and imposes a huge economic burden on their families and society. There are approximately 2 million patients worldwide, and the annual related costs exceed US$24 billion. The development of new drugs to treat MS is urgent and arduous.

[0004] For the treatment of MS, the clinical drug Fingolimod (FTY-720) was developed by Japanese scientists in the late 1980s by isolating the fungus Cordyceps sinensis and its close relative Cordyceps sinensis as research objects to obtain the compound ISP-I with strong immunosuppressive activity, and finally obtained it as a lead compound through a series of structural optimization. It is the first oral drug approved by the FDA for the treatment of RRMS; dimethyl fumarate was originally a common industrial raw material for antibacterial, moisture-proof and mildew-proofing. It is inexpensive and has a simple structure. It was successfully developed by Biogen Idec, a leader in the field of multiple sclerosis, for the treatment of multiple sclerosis. It was approved for marketing by the US FDA in 2013 and swept the world like a blockbuster after its launch; on July 23, 2018, Aubagio (teriflunomide tablets) developed by Sanofi was approved by the National Medical Products Administration for marketing in China for the treatment of relapsing multiple sclerosis. Fingolimod, dimethyl fumarate and teriflunomide, three clinical drugs for MS, are all small molecule compounds. Their successful development has also inspired many scientists to turn their attention to natural small molecule compounds, hoping to discover drugs that can cure MS.

[0005] Lawsone is a natural small molecule compound that can react with keratin to produce permanent dyeing. It has been used as a natural dye for dyeing hair and nails and tattoos for more than 5,000 years and has been approved by the FDA for cosmetic use. However, to date, relevant literature reports have mostly focused on the extraction, content determination, chemical synthesis, structural modification and antibacterial properties of Lawsone and its derivatives, and there are almost no literature reports related to immunity.

[0006] Furthermore, patent document WO1991006863A1 discloses that dichloroallyl lawsone can treat MS, and patent US20030139353A1 discloses that topoisomerase inhibitors can treat MS, but the patent clearly states that plumbagin, lawsone or juglone have no effect on the relief of MS. Summary of the invention

[0007] In order to further study the pathogenic mechanism of MS and develop more natural small molecule compounds that can cure MS. This application provides the use of Lawsone in the treatment of autoimmune diseases (especially MS or EAE), and experimental verification shows that Lawsone administration not only delays the onset of EAE in mice, but also alleviates the severity of the disease in a dose-dependent manner, and is safe and has no toxic side effects. It shows that Lawsone can significantly alleviate the clinical symptoms of EAE in multiple sclerosis (MS) animal model mice, and both in vivo and in vitro results show significant inhibition of inflammatory CD4 + Furthermore, the present application clarifies from the mechanism of action that the therapeutic effect of Lawsone on autoimmune diseases is mainly reflected in the inhibition of inflammatory CD4 + It inhibits the differentiation of T cell subtype Th1 (both in vivo and in vitro), and significantly reduces the Th1 cells in the spleen and draining lymph nodes of the peripheral immune system and infiltrating into the brain and spinal cord of the central nervous system in vivo, but does not affect Th17 and Treg cells.

[0008] In fact, there is evidence that both Th1 and Th17 cells can independently induce EAE. Based on the differences in tissue morphology and CNS chemokine spectra, it is inferred that Th1 cells and Th17 cells may induce different types of EAE through different mechanisms. In other words, the present application provides a more thorough understanding of the therapeutic targets of Lawsone from a mechanistic perspective, providing new ideas for the treatment of autoimmune diseases. It also further proves that Lawsone's treatment of autoimmune diseases is mainly reflected in the treatment of autoimmune diseases mediated by Th1, and has no effect on Th17-mediated autoimmune diseases. The differences and commonalities between Th1 and Th17 cells in the pathogenesis of EAE will provide effective ideas and targets for the treatment of MS.

[0009] The first aspect of the present invention provides a use of an Impatiens balsamina extract in preparing a medicament for treating autoimmune diseases.

[0010] Preferably, the autoimmune disease is an organ-specific autoimmune disease or a systemic autoimmune disease. The organ-specific autoimmune disease includes, but is not limited to, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple cerebrospinal sclerosis, and acute idiopathic polyneuritis. The systemic autoimmune disease includes, but is not limited to, systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, pemphigus, dermatomyositis, mixed connective tissue disease, autoimmune hemolytic anemia, thyroid autoimmune disease, and ulcerative colitis.

[0011] Further preferably, the autoimmune disease includes but is not limited to allergy, autoimmune encephalomyelitis, multiple sclerosis, asthma, dermatitis, myocarditis, nephritis, hepatitis, systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, ulcerative colitis, autoimmune liver disease, diabetes, pain or neurological disorders.

[0012] In a specific embodiment of the present invention, the autoimmune disease is autoimmune encephalomyelitis (experimental autoimmune encephalomyelitis (EAE)) or multiple sclerosis (MS).

[0013] The second aspect of the present invention provides a method for inhibiting CD4 + T cell subtype ratio or its application in myelin regeneration.

[0014] The third aspect of the present invention provides a method for preparing a method for inhibiting CD4 + Application of T cell subtype ratio in the preparation of myelin regeneration products.

[0015] Preferably, the CD4 + The T cell subtype is selected from Th1, Th17 or Treg.

[0016] Preferably, the Impatiens balsamina extract is selected from extracts of roots, stems, leaves, flowers, fruits or seeds of Impatiens balsamina.

[0017] In a specific embodiment of the present invention, the Impatiens balsamina extract is an extract from the roots of Impatiens balsamina.

[0018] The Impatiens balsamina extract can be extracted by any conventional method, preferably by decoction, percolation, ultrasonic extraction, immersion, steam distillation or supercritical extraction.

[0019] Preferably, the Impatiens balsamina extract contains 50%-99.9% Lawsone by mass, and specifically can be 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% Lawsone.

[0020] Further preferably, the Impatiens balsamina extract may also contain flavonols, 2-methoxy-1,4-naphthoquinone, kaempferol-3-rhamnosyl diglucoside, 2-hydroxy-1,4-naphthol, scopoletin, isoflurane or spinasterol.

[0021] A fourth aspect of the present invention provides a drug for treating autoimmune diseases, wherein the drug comprises Impatiens balsamina extract or Lawsone.

[0022] Preferably, the drug further comprises pharmaceutically acceptable excipients, including but not limited to carriers, diluents, adhesives, lubricants or wetting agents.

[0023] Preferably, the pharmaceutical dosage form can be tablets, capsules, pills, injections, inhalants, lozenges, suppositories, emulsions, microemulsions, submicroemulsions, nanoparticles, gels, powders, suspensoids, creams, jellies or sprays, etc.

[0024] Preferably, the drug can be administered orally, enterally, subcutaneously, intramuscularly, intravenously, nasally, transdermally, subconjunctivally, intraocularly, orbitally, retro-ocularly, retinaly, choroidally, intrathecally, etc.

[0025] The fifth aspect of the present invention provides the use of Lawsone in the preparation of a medicament for treating autoimmune diseases.

[0026] Preferably, the autoimmune disease is an organ-specific autoimmune disease or a systemic autoimmune disease. The organ-specific autoimmune disease includes, but is not limited to, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple cerebrospinal sclerosis, and acute idiopathic polyneuritis. The systemic autoimmune disease includes, but is not limited to, systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, pemphigus, dermatomyositis, mixed connective tissue disease, autoimmune hemolytic anemia, thyroid autoimmune disease, and ulcerative colitis.

[0027] Further preferably, the autoimmune disease includes but is not limited to allergy, autoimmune encephalomyelitis, multiple sclerosis, asthma, dermatitis, myocarditis, nephritis, hepatitis, systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, ulcerative colitis, autoimmune liver disease, diabetes, pain or neurological disorders.

[0028] In a specific embodiment of the present invention, the autoimmune disease is autoimmune encephalomyelitis (experimental autoimmune encephalomyelitis (EAE)) or multiple sclerosis (MS).

[0029] The sixth aspect of the present invention provides the effect of Lawsone on inhibiting CD4 + Application of T cell subtype ratio in myelin regeneration.

[0030] In a seventh aspect of the present invention, there is provided a method for preparing a method for inhibiting CD4 + Application of T cell subtype ratio in the preparation of myelin regeneration products.

[0031] Preferably, the CD4 + The T cell subtype is selected from Th1, Th17 or Treg.

[0032] The eighth aspect of the present invention provides a method for treating autoimmune diseases, wherein the method comprises administering Impatiens balsamina extract, Lawsone or the above-mentioned drugs to an individual.

[0033] The ninth aspect of the present invention provides a method for analyzing the pathogenic mechanism of an autoimmune disease or determining a target for treatment of an autoimmune disease, the method comprising applying Impatiens balsamina extract or Lawsone or the above-mentioned drugs to an individual, and then detecting potential targets.

[0034] Preferably, the detection method comprises RNA-seq transcriptome and protein spectrum analysis.

[0035] Preferably, the targets include targets at the gene level and protein level. Further preferably, the targets at the gene level are found using CRISPR / Cas9 technology, and the targets at the protein level are found using DARTS-MS technology.

[0036] The tenth aspect of the present invention provides a method for constructing an animal model, which includes the steps of determining the target of the autoimmune disease to be treated, and then constructing the animal model by combining inhibitors or agonists, knocking out or overexpressing the target gene, etc.

[0037] The "individual" described in the present invention can be a human or a non-human animal. The non-human animal is preferably a non-human mammal, including but not limited to monkeys, dogs, rabbits, mice, cattle, sheep, etc.

[0038] The term "pharmaceutically acceptable" as used herein refers to a substance that neither significantly stimulates the organism nor inhibits the biological activity and properties of the active substance of the administered product. The term "pharmaceutically acceptable excipient" includes, but is not limited to, carriers, diluents, adhesives, lubricants, and wetting agents.

[0039] The "application" mentioned in the present invention can be oral administration, enteral administration, subcutaneous injection, intramuscular injection, intravenous injection, nasal administration, transdermal administration, subconjunctival administration, intraocular administration, orbital administration, retro-ocular administration, retinal administration, choroidal administration, intrathecal injection, etc.

[0040] The "CD4 + The proportion of T cell subtypes is CD4 + The number of each subtype of T cells accounts for the total CD4 + In one embodiment of the present invention, the CD4 + The T cell subtype is selected from Th1, Th17 or Treg. That is, the Th1 ratio is the number of Th1 cells to CD4 + The ratio of T cells. The same is true for the ratio of Th17 or Treg.

[0041] The term "inhibition" as used herein refers to, for example, inhibition of CD4 + The proportion of T cell subtypes, that is, when other conditions remain unchanged, the application of a certain compound reduces the original proportion compared with the non-application of a certain compound.

[0042] The "Lawsone" mentioned in this application is hyaluronan, hyaluronan, quinone anthraquinone xanthones or henna, and the chemical formula is: C 10 H6O3, the structural formula is:

[0043]

[0044] As used herein, "treating" means slowing, interrupting, preventing, controlling, stopping, alleviating, or reversing the progression or severity of a sign, symptom, disorder, condition, or disease after the disease has begun to develop, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, conditions, or disorders. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

[0046] Figure 1: CD4 + Flow cytometry of T cell differentiation into Th1 cells in vitro ( Figure 1A ) and statistical graphs ( Figure 1B ), *p<0.05, ***p<0.001 (Student's t-test).

[0047] Figure 2 :The results of Lawsone differentiation into Th17 and Treg, where Figure A represents CD4 + Flow cytometry analysis of T cells differentiating into Th17 cells in vitro; Figure B represents the percentage of Th17 cells in CD4 + T cell ratio statistics; Figure C represents CD4 + Flow cytometry analysis of T cells differentiated into Treg cells in vitro; Figure D represents the percentage of Treg cells in CD4 + T cell ratio statistics.

[0048] Figure 3: Th1 ( Figure 3A )、Th17( Figure 3B ) and Treg( Figure 3C ) cells account for CD4 + The proportion of T cells.

[0049] Figure 4 :Lawsone reduces CD4 in spleen and draining lymph nodes in EAE mice + The proportion of T cell subsets Th1, where A is the proportion of Th1, Th17 and Treg cells in the spleen CD4 + The proportion of T cells; B is the proportion of Th1, Th17 and Treg cells in the draining lymph nodes + Proportion of T cells, *p<0.05, ***p<0.001 (Student's t-test).

[0050] Figure 5 : SDS-PAGE gel silver staining.

[0051] Figure 6 :Lawsone alleviates the clinical symptoms of EAE in mice by inhibiting Th1 cells, wherein Figure A represents the low-dose Lawsone (3 mg / Kg) group; Figure B represents the medium-dose Lawsone (10 mg / Kg) group; Figure C represents the high-dose Lawsone (30 mg / Kg) group.

[0052] Figure 7 :Therapeutic administration of Lawsone significantly improved the disease progression and severity of EAE mice. Figure A shows oral administration starting from the 14th day after immune induction of the EAE model, and Figure B shows oral administration starting from the 18th day after immune induction of the EAE model.

[0053] Figure 8 :The effect of Lawsone on immune homeostasis in mice. A is the change in body weight; B is the change in the main immune cell types in the spleen; C is the change in the main immune cell types in the draining lymph nodes; D is the change in the main immune cell types in the blood. DETAILED DESCRIPTION

[0054] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0055] Preparation of the disease mouse model (EAE mouse model) used in the examples:

[0056] 8-10 week old male C57BL / 6 mice were selected and 200 μg oligodendrocyte myelin glycoprotein MOG was used to 35-55 The mice were immunized subcutaneously on the back with an equal volume of complete Freund's adjuvant (containing 5 mg / ml tuberculin MTB), and 200 ng of pertussis toxin PTX was injected intraperitoneally on day 0 and day 2 to obtain the EAE mouse model.

[0057] Example 1 Lawsone inhibits Th1 differentiation in vitro

[0058] This example mainly determines whether Lawsone directly affects pathogenic CD4 + The differentiation of Th1 T cell subsets is not affected.

[0059] Test steps:

[0060] The spleen of 7-8 week old C57BL / 6 mice was isolated, red blood cells were removed with red blood cell lysis buffer, and CD4 + T cells were activated by adding CD3 and CD28 antibodies, and the corresponding cytokines and neutralizing antibodies were added to induce Th1 (IL-12 and anti-IL4), Th17 (TGFβ, IL-6, TNFα, IL-1β, IL-23, anti-IL4 and anti-IFNγ) or Treg (TGFβ, IL-2 and anti-IFNγ) differentiation. At the same time, Lawsone was added at a final concentration of 25μM, 50μM or 100μM, and 3 replicates were set for each concentration gradient. The same volume of dimethyl sulfoxide (DMSO) was added to the control group, and the cells were cultured in a cell culture incubator at 37℃ and 5% CO2 for 72h. The effects of drugs on cell growth status were observed under a microscope, and the expression of intracellular Th1 characteristic cytokine IFN-γ, as well as the changes in the expression of Th17 and Treg characteristic cytokines intracellular IL-17a and nuclear Foxp3 were analyzed by flow cytometry.

[0061] Test results:

[0062] The results of Lawsone on Th1 differentiation are shown in Figure 1, which show that Lawsone inhibits Th1 differentiation in a dose-dependent manner without affecting the cell number, growth status and clone formation ability.

[0063] The results of Lawsone on Th17 and Treg differentiation are shown in Figure 2 The results showed that Lawsone did not affect Th17 and Treg differentiation in vitro.

[0064] Example 2 Lawsone reduces CD4 in EAE mice + Proportion of Th1 T cell subsets

[0065] From the third day of EAE mouse modeling, different doses of Lawsone or solvent were administered orally once a day. 10% chloral hydrate anesthesia was performed, and the immune peripheral organs spleen and draining lymph nodes of EAE mice in the solvent control group (DMSO) and Lawsone-treated group were collected at the pre-onset (7th day) and the central immune system brain and spinal cord at the peak of onset (19th day). Single cell suspensions were obtained and then MOG 35-55 Antigen re-stimulation, CD4 + The staining of T subsets Th1, Th17 and Treg cells was performed, and flow cytometry was used to analyze the effects of Lawsone administration on Th1, Th17 and Treg cells in EAE mice.

[0066] On the 19th day of EAE induction, the infiltrating immune cells in the brain and spinal cord of mice in the control group and the Lawsone-treated group were collected, and the flow cytometry analysis results are shown in Figure 3. Compared with the control group, Lawsone-treated mice not only significantly reduced the proportion of Th1 cells infiltrating into the brain and spinal cord of the central nervous system, but also had no significant changes in the proportions of Th17 and Treg cells.

[0067] Immune cells in the spleen and draining lymph nodes of mice on day 7 after EAE induction were detected by staining of cell surface antigen CD4, intracellular IFN-γ, IL17A and nuclear Foxp3. Flow cytometry was used to detect the expression of splenic CD4 + The results of the proportion of Th1, Th17 and Treg cell subsets in T cells are shown in Figure 4 The results showed that Th1 cells in the spleen and draining lymph nodes were significantly reduced.

[0068] Example 3 Lawsone Target

[0069] CD4 T cells were isolated from spleens of 7-8 week old C57BL / 6 mice. +T cells were induced to polarize to Th1 in vitro by adding Lawsone or DMSO at a final concentration of 100 μM and inducing differentiation for 72 h. Th1 cells treated with DMSO (control group) or Lawsone were collected and subjected to DARTS experiment. The differentially expressed proteins protected by Lawsone treatment were identified by silver staining after SDS-PAGE electrophoresis.

[0070] After 72 h, cell lysate proteins were collected and added at a ratio of pronase mass to total protein mass = 1:100, 1:500 or 1:1000. After incubation at room temperature for 20 min, the reaction was terminated by protease inhibitors. The results of silver staining after SDS-PAGE electrophoresis were shown in Figure 2. Figure 5 The results showed that compared with the control group, protein bands that were not reduced due to binding with Lawsone were shown near 40kD, 50kD and 70kD. The differential bands were cut out for subsequent mass spectrometry detection to determine the target of Lawsone.

[0071] Draining lymph nodes, spleen, peripheral blood in the early stage of EAE, and brain and spinal cord in the peak stage of EAE were collected, and barcoding was performed on each sample. The cells were resuspended in 50 μl Maxpar cell staining buffer, Fc-receptor (BioLegend) blocking solution was added, incubated for 10 min, and then 50 μl of heavy metal element labeled antibody mixture was added to make the total volume 100 μl for cell staining, including 18 cell surface markers, 10 phosphorylated proteins, 7 transcription factors and other inflammatory marker antibodies. The list of target antibodies detected by CyTOF experiment is detailed in Table 1-3.

[0072] Table 1

[0073]

[0074]

[0075] Table 2

[0076]

[0077] Table 3

[0078]

[0079] Example 4 Lawsone significantly alleviates clinical symptoms in EAE mice

[0080] Preventive oral administration began on the third day after the induction of the EAE model, and the mice were divided into a low-dose group of Lawsone (3 mg / Kg), a medium-dose group of Lawsone (10 mg / Kg), and a high-dose group of Lawsone (30 mg / Kg). Each mouse was given 200 μl of the drug at the same time every day, and the control group was given the same volume of solvent (DMSO) control. The clinical scores of EAE mice were recorded every day after the onset of the disease. The neurological function of mice was scored using the standard 5-point method: 0 points, no clinical manifestations; 1 point, paralysis of the tail; 2 points, mild paresis (weakness, incomplete paralysis of one or both hind limbs); 3 points, paraplegia (complete paralysis of both hind limbs); 4 points, paraplegia with forelimb paralysis; 5 points, dying or death.

[0081] The results showed that the administration of Lawsone not only delayed the onset of EAE in mice, but also alleviated the severity of EAE in a dose-dependent manner (see Figure 6 ).

[0082] Example 5 Therapeutic administration of Lawsone significantly improves the disease progression and severity of EAE mice

[0083] Considering that MS patients generally receive drug treatment only after showing clinical symptoms, this example administers therapeutic drugs to EAE mice at different stages of the disease process.

[0084] The treatment was started by intragastric administration on the 14th day (acute progression, average clinical score was 1.6) and the 18th day (peak period) after EAE immune induction, with a dose of 30 mg / kg. Each mouse was given 200 μl at the same time every day, and the same volume of solvent (DMSO) was used as a control. The clinical score was recorded every day. The results showed that Lawsone can effectively block the rapid development of the disease and significantly alleviate the severity of the disease in EAE mice (see Figure 7 ), indicating that Lawsone not only intervenes in the pathological activities of EAE at the onset stage, but also has a significant effect on the treatment of the disease once it occurs.

[0085] Example 6 Lawsone does not affect the immune homeostasis of mice

[0086] Lawsone has the most significant effect at a dose of 30 mg / Kg. The purpose of this example is to verify the safety at this dose.

[0087] 7-8 week old C57BL / 6 mice were selected and given 30 mg / kg Lawsone by oral gavage every day. Each mouse was given 200 μl per day. The control group was given the same volume of solvent control. The body weight was measured every 5 days. After 20 days, the peripheral spleen, draining lymph nodes and blood were taken respectively. The changes of different immune cell types were analyzed by flow cytometry. The results showed that Lawsone did not affect the peripheral spleen, draining lymph nodes and CD4 +T, CD8 + T, CD19, CD11b, NK1.1, Ly6G and Ly6C immune cell subsets (see Figure 8 ), the mice grew and developed normally, indicating that this dose did not affect the immune homeostasis of mice and had no toxic side effects.

[0088] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0089] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. The use of Lawsone in the preparation of a drug for treating autoimmune diseases, characterized in that: The autoimmune disease is autoimmune encephalomyelitis or multiple sclerosis mediated by Th1.

2. The use according to claim 1, characterized in that: Lawsone inhibits CD4 + Differentiation of T cells to Th1 subtype for the treatment of autoimmune diseases.

3. The use according to claim 2, characterized in that: Lawsone administration significantly reduced the proportion of Th1 cells infiltrating into the brain and spinal cord of the central nervous system, while the proportions of Th17 and Treg cells did not change significantly.

4. The use according to any one of claims 1 to 3, characterized in that: The medicine comprises Lawsone and pharmaceutically acceptable excipients.

5. The use according to claim 4, characterized in that: The auxiliary material is selected from a carrier, a diluent, a binder, a lubricant or a wetting agent.

6. The use according to claim 1, characterized in that: The dosage form of the drug is tablets, capsules, pills, injections, inhalants, lozenges, suppositories, emulsions, microemulsions, submicroemulsions, nanoparticles, gels, powders, suspensions, creams, jellies or sprays.

Images