Application of alpha-ketoglutaric acid in preparation of medicine for treating systemic lupus erythematosus

Abstract

The invention relates to the technical field of medicines, in particular to application of alpha-ketoglutaric acid in preparation of a medicine for treating systemic lupus erythematosus. According to the application, the alpha-ketoglutaric acid shows certain potential in treatment of systemic lupus erythematosus for the first time, and specifically, the alpha-ketoglutaric acid can relieve spleen swelling and reduce the weight of the spleen; the kidney injury is relieved, and the deposition of IgG and IgM in the glomerulus is reduced; and meanwhile, the activation of B cells and the differentiation of hair growth center B cells and plasma cells are inhibited. The alpha-ketoglutaric acid provided by the invention can relieve systemic lupus erythematosus, can effectively avoid dependence and long-term side effects of traditional medicines for treating systemic lupus erythematosus, and provides a brand new experimental basis for developing safer and more effective treatment means for systemic lupus erythematosus.

Description

Technical Field

[0001] The invention relates to the technical field of medicines, and in particular to application of alpha-ketoglutaric acid in preparing medicines for treating systemic lupus erythematosus. Background Art

[0002] Systemic lupus erythematosus (SLE) is a chronic autoimmune connective tissue disease characterized by a long course and recurrent attacks, accompanied by the production of a large number of autoantibodies and inflammatory damage to multiple organs. There are more than 1 million SLE patients in my country, mainly in women of childbearing age between 20 and 40 years old. The specific pathogenesis of the disease has not yet been fully clarified, and the clinical manifestations are complex and diverse. Among them, the butterfly-shaped erythema that appears on the bridge of the nose and cheeks is a hallmark of SLE. In addition, due to the extensive attack of the immune system on its own healthy tissues, SLE can lead to the involvement of multiple key organs and systems, such as lupus nephritis, neuropsychiatric lupus, blood system abnormalities and joint damage. Therefore, it is crucial for SLE patients to develop a reasonable and targeted treatment plan.

[0003] At present, the treatment strategy of SLE mainly focuses on controlling disease activity and relieving clinical symptoms. Commonly used therapeutic drugs include glucocorticoids, antimalarial drugs (such as chloroquine), immunosuppressants (such as cyclophosphamide, mycophenolate mofetil) and biological agents (such as belimumab, rituximab). Although these drugs can effectively relieve the condition of most patients, they are often accompanied by drug dependence and a series of adverse reactions. For example, glucocorticoids can quickly control inflammatory responses, but long-term use may lead to serious complications such as infection and osteoporosis; immunosuppressants are used in critically ill patients or to prevent relapse during hormone reduction, but they may also cause side effects such as bone marrow suppression and gonadal suppression.

[0004] Therefore, there is an urgent need to develop drugs for the treatment of systemic lupus erythematosus with higher targeting and lower toxicity and side effects, so as to effectively improve the quality of life of patients and improve the problems of large side effects and strong dependence of drugs in long-term drug treatment. Summary of the invention

[0005] In order to develop a new type of therapeutic drug with high targeting and low toxicity and side effects, the present invention provides the use of α-ketoglutaric acid in the preparation of a drug for treating systemic lupus erythematosus. The α-ketoglutaric acid provided by the present invention can relieve systemic lupus erythematosus, can effectively avoid the dependence and long-term side effects of traditional therapeutic drugs, and provides a new experimental basis for the development of safer and more effective treatments for systemic lupus erythematosus.

[0006] The invention provides an application of alpha-ketoglutaric acid in preparing a medicine for treating systemic lupus erythematosus.

[0007] Furthermore, the α-ketoglutaric acid is used to prepare a drug for alleviating spleen enlargement.

[0008] Furthermore, the α-ketoglutaric acid is used to prepare a drug for alleviating kidney damage.

[0009] Furthermore, the α-ketoglutaric acid is used to prepare a drug for reducing IgG and / or IgM deposition in glomeruli.

[0010] Furthermore, the drug has α-ketoglutaric acid as the only active ingredient.

[0011] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0012] Furthermore, the medicine is an oral preparation or an injection.

[0013] Furthermore, the oral preparation is any one of tablets, capsules, pills, powders, granules and syrups.

[0014] Furthermore, the injection is an injection solution or sterile powder for injection.

[0015] Furthermore, the unit preparation of the injection is 1 mL; the injection is prepared by the following steps: After 10 mg of α-ketoglutaric acid, 100 μL of physiological saline and 400 μL of polyethylene glycol 300 are mixed, 50 μL of Tween 80 is added and mixed, and the volume is made up to 1 mL with physiological saline to obtain the injection.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The present invention shows for the first time that α-ketoglutarate has certain potential in the treatment of lupus erythematosus, which is specifically manifested in that α-ketoglutarate can reduce splenomegaly and spleen weight; at the same time, it can reduce kidney damage, reduce the deposition of IgG and IgM in the glomeruli, inhibit the activation of B cells and the differentiation of B cells and plasma cells in the germinal center, effectively treat systemic lupus erythematosus, avoid the dependence and long-term side effects of traditional therapeutic drugs, and provide a new experimental basis for the development of safer and more effective treatments for systemic lupus erythematosus.

[0017] Compared with the current conventional glucocorticoid and immunosuppressant treatments for systemic lupus erythematosus, α-ketoglutarate is more targeted. In addition, since α-ketoglutarate is a metabolite contained in the human body, the side effects are relatively small. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 The level of α-ketoglutarate in serum and MPL / lpr Figure 3 shows the content of α-ketoglutarate in the serum of spontaneous lupus mice and imiquimod (IMQ)-induced lupus mice; Figure a shows the categories and percentages of 100 organic acid metabolites detected by organic acid targeted metabolomics; Figure b shows the average content of organic acid metabolites such as α-ketoglutarate in the serum of healthy control group (n=9) and SLE patient group (n=9), P value and the changes in organic acid content in SLE patients compared with healthy people; Figure c shows the analysis results of the content of α-ketoglutarate in the peripheral blood serum of 26 healthy people, 15 inactive SLE patients and 54 active SLE patients; Figure d shows the results of the determination of control mice (n=11) and MRL / lpr Figure 5 shows the analysis results of α-ketoglutarate content in the serum of mice (n=10); Figure e shows the analysis results of α-ketoglutarate content in the serum of control mice (n=15) and IMQ-induced lupus mice (n=14); * indicates significant difference between groups, the more * the more significant the difference, ns indicates no difference.

[0020] Figure 2 For α-ketoglutarate in the treatment of MRL / lpr Study on spontaneous lupus mice; Figure, a shows the MRL / lpr Representative images of the spleen of lupus mice; (b) shows the MRL / lpr Spleen weight of lupus mice; c shows the MRL / lpr Anti-dsDNA antibody levels in the serum of lupus mice; d is the MRL / lpr The results of H&E staining of the kidneys of lupus mice. The lower row of pictures are the enlarged pictures in the black boxes corresponding to the upper row of pictures; Figure e is the MRL / lprIgG and IgM immunofluorescence of lupus mice. The upper row of pictures shows IgG immunofluorescence, and the lower row of pictures shows IgM immunofluorescence. The image scale is 100 μm. * indicates significant differences between groups.

[0021] Figure 3 For α-ketoglutarate in the treatment of MRL / lpr A study in spontaneous lupus mice; a shows the MRL / lpr B220 in the spleen of lupus mice + B cell CD86 activation; c is the MRL / lpr B220 in the lymph nodes of lupus mice + B cell CD86 activation; Figure e shows MRL / lpr Germinal center B cells (B220) in the splenic lymph nodes of lupus mice + , GL7 + g shows the proportion of MRL / lpr Germinal center B cells (B220) in lymph nodes of lupus mice + , GL7 + Figure i shows the MRL / lpr Plasma cells in the spleen of lupus mice (B220 + , CD138 + Figure k shows the proportion of MRL / lpr Plasma cells in lymph nodes of lupus mice (B220 + , CD138 + ), and Figures b, d, f, h, j, and l are the statistical result graphs corresponding to Figures a, c, e, g, i, and k respectively; * indicates that there are significant differences between groups, and the more *s there are, the more significant the differences are.

[0022] Figure 4This is a study on the effect of α-ketoglutarate in the treatment of lupus mice induced by imiquimod (IMQ); wherein, Figure a shows the disease treatment progress of IMQ-induced lupus mice after control treatment or injection of α-ketoglutarate (5 μL of α-ketoglutarate preparation per gram of body weight, i.e., 50 μg / g; 10 μL of α-ketoglutarate preparation per gram of body weight, i.e., 100 μg / g); Figure b shows a representative picture of the spleen of IMQ-induced lupus mice after control treatment or injection of α-ketoglutarate (50 μg / g, 100 μg / g); Figure c shows the spleen weight of IMQ-induced lupus mice after control treatment or injection of α-ketoglutarate (50 μg / g, 100 μg / g); Figure d shows the spleen weight of IMQ-induced lupus mice after control treatment or injection of α-ketoglutarate (50 μg / g, 100 μg / g); Figure 4 shows the levels of anti-dsDNA antibodies in the serum of IMQ-induced lupus mice after treatment with control agent or injection of α-ketoglutarate (50 μg / g, 100 μg / g); Figure e shows the results of H&E staining of the kidneys of IMQ-induced lupus mice after control agent treatment or injection of α-ketoglutarate (50 μg / g, 100 μg / g), and the lower row of pictures are the enlarged pictures in the black boxes corresponding to the upper row of pictures; Figure f shows the IgG and IgM immunofluorescence of IMQ-induced lupus mice after treatment with control agent or injection of α-ketoglutarate (50 μg / g, 100 μg / g), the upper row of pictures are IgG immunofluorescence, and the lower row of pictures are IgM immunofluorescence, and the scale bars are 100 μm; * indicates that there are significant differences between groups, and the more *s there are, the more significant the differences are.

[0023] Figure 5 This is a study on the effect of α-ketoglutarate in treating Imiquimod (IMQ)-induced lupus mice; Figure a shows B220 in the spleen of IMQ-induced lupus mice after control treatment or injection of α-ketoglutarate (50 μg / g, 100 μg / g). + B cell CD86 activation; b shows B220 in the spleen of IMQ-induced lupus mice after control treatment or injection of α-ketoglutarate (50 μg / g, 100 μg / g) + B cell CD86 activation statistics; Figure c shows the germinal center B cells (B220) in the splenic lymph nodes of IMQ-induced lupus mice after control treatment or injection of α-ketoglutarate (50 μg / g, 100 μg / g). + , GL7 + Figure d shows the proportion of plasma cells (B220, CD95) in the spleen of lupus mice induced by IMQ after treatment with control or injection of α-ketoglutarate (50 μg / g, 100 μg / g). + , CD138 + ) proportion; Figure e is the statistical chart corresponding to Figure c; Figure f is the statistical chart corresponding to Figure d; * indicates that there are significant differences between groups, and the more * there are, the more significant the difference is.

[0024] Figure 6 Figure 2 is the effect of α-ketoglutarate on B cell activation mediated by TOLL-like receptor (TLR); Figure a shows the B220 in LPS after α-ketoglutarate treatment + CD86 statistical graph; Figure b shows B220 in LPS after α-ketoglutarate treatment + CD40 statistical chart; Figure c shows B220 in R848 after α-ketoglutarate treatment + CD86 statistical graph; Figure d shows B220 in R848 after α-ketoglutarate treatment + CD40 statistical graph; Figure e shows B220 stimulated by CpG1826 after α-ketoglutarate treatment + B cell CD86 statistics; Figure f shows B220 stimulated by CpG1826 after α-ketoglutarate treatment + B cell CD40 statistics; g is B220 + B cell TNF-α mRNA expression level; h is B220 + Figure i shows the expression level of IL-12 mRNA in B cells; Figure i shows the phosphorylation expression levels of p38, Erk, JNK, and p65 detected by Western blot after α-ketoglutarate treatment for 30 min and 60 min, with β-actin as a reference; * indicates significant differences between groups, the more * the more significant the difference, and ns indicates no difference. DETAILED DESCRIPTION

[0025] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0026] The current treatment strategy for systemic lupus erythematosus mainly focuses on controlling disease activity and relieving clinical symptoms. Commonly used therapeutic drugs include glucocorticoids, antimalarial drugs (such as chloroquine), immunosuppressants (such as cyclophosphamide, mycophenolate mofetil) and biological agents (such as belimumab, rituximab), etc. Although these drugs can effectively alleviate the condition of most patients, they are often accompanied by drug dependence and a series of adverse reactions. Therefore, there is an urgent need to develop drugs with higher targeting and low toxicity for the treatment of systemic lupus erythematosus, so as to effectively improve the quality of life of patients and improve the problems of large side effects and strong dependence of drugs in long-term drug treatment.

[0027] The present invention provides the use of α-ketoglutaric acid in the preparation of drugs for treating systemic lupus erythematosus. The present invention firstly finds that the content of α-ketoglutaric acid in the serum of patients with systemic lupus erythematosus is significantly higher than that of healthy controls through organic acid targeted metabolomics analysis, and then constructs a spontaneous lupus erythematosus animal model and an induced lupus erythematosus animal model, and finds that the α-ketoglutaric acid provided by α-ketoglutaric acid can alleviate systemic lupus erythematosus, which is manifested in that α-ketoglutaric acid can reduce splenomegaly and spleen weight; reduce kidney damage and reduce the deposition of IgG and IgM in glomeruli; and inhibit the activation of B cells and the differentiation of germinal center B cells and plasma cells. It can effectively avoid the dependence and long-term side effects of traditional therapeutic drugs, and provide a new experimental basis for the development of safer and more effective treatments for systemic lupus erythematosus.

[0028] Example 1: Preparation method of α-ketoglutaric acid solution After evenly mixing 10 mg of α-ketoglutaric acid, 100 μL of physiological saline and 400 μL of polyethylene glycol 300, add 50 μL of Tween 80 and mix evenly, and make up to 1 mL with physiological saline to obtain a 1% α-ketoglutaric acid solution.

[0029] Example 2: Validation of α-ketoglutarate in the treatment of lupus erythematosus 1. Materials and Methods 1. Materials and reagents 57BL / 6 mice, 6-8 weeks old, were purchased from Pengyue Experimental Animal Breeding Co., Ltd., Jinan, China; MRL / lpr mice, 8 weeks old, were purchased from Jiangsu Ailingfei Biotechnology Co., Ltd.; Imiquimod cream (IMQ) was purchased from Sichuan Mingxin Pharmaceutical Co., Ltd., TLR4 agonist lipopolysaccharide (LPS) was purchased from Shanghai Bio-Technology Co., Ltd., TLR7 agonist resiquimod (R848) was purchased from GlpBIO, USA, and TLR9 agonist CpG (deoxyribonucleic acid chain with a sequence length of 20) was synthesized by Sangon Biotechnology (Shanghai) Co., Ltd.; α-ketoglutarate content detection kit was purchased from Beijing Solebold Technology Co., Ltd., anti-dsDNA antibody detection kit was purchased from Bethyl Laboratories, USA, and human lymphocyte separation medium Ficoll-Paque™ PLUS was purchased from GE Healthcare, USA.

[0030] The following antibodies used for flow cytometry were purchased from BioLegend, USA: FITC-labeled anti-mouse B220 antibody, PE-labeled anti-mouse GL7 antibody, PE-labeled anti-mouse CD40 antibody, APC-labeled anti-mouse CD86 antibody, APC-labeled anti-mouse CD138 antibody, APC-labeled anti-mouse CD95 antibody. The following antibodies used for western blot were purchased from Cell Signaling Technology, USA: anti-Erk antibody, anti-p-Erk antibody, anti-JNK antibody, anti-p-JNK antibody, anti-p38 antibody, anti-p-p38 antibody, anti-p65 antibody, anti-p-p65; The following antibodies used for western blot were purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.: anti-β-actin antibody, HRP-labeled Goat Anti-Rabbit IgG antibody, HRP-labeled Goat Anti-Mouse IgG antibody. The following antibodies used for immunofluorescence were purchased from Invitrogen, USA: anti-mouse IgG antibody labeled with fluorescein Alexa Fluor 488, and anti-mouse IgM antibody labeled with fluorescein Alexa Fluor 488.

[0031] 2. Methods 2.1 Construction of mouse model Construction of spontaneous lupus erythematosus mouse model: To investigate the effect of α-ketoglutarate on MRL / lpr To investigate the effect of morbidity on mice, 12 10-week-old female MRL / lpr The mice were randomly divided into two groups. One group was treated with intraperitoneal injection of PBS as a control group, and the other group was treated with intraperitoneal injection of α-ketoglutarate (75 μg / g) twice a week for a total of 6 weeks.

[0032] Construction of induced lupus erythematosus mouse model: In order to construct an imiquimod cream (IMQ)-induced lupus mouse model, six 8-week-old female C57 BL / 6 mice were selected, and 1.25 mg of 5% imiquimod cream (IMQ) was applied to their right ears three times a week. After 10 weeks of treatment, the mice were killed by CO2 inhalation, and the relevant indicators in the spleen and lymph nodes of the mice were detected.

[0033] To study the effect of α-ketoglutarate on IMQ-induced lupus mice, 30 female C57BL / 6 mice were randomly divided into 5 groups: Group A was treated with intraperitoneal injection of 200 μL PBS only as a control, Group B was treated with intraperitoneal injection of 200 μL α-ketoglutarate (100 μg / g) only as a control, Group C was treated with IMQ and intraperitoneal injection of 200 μL PBS, Group D was treated with IMQ and intraperitoneal injection of 100 μL α-ketoglutarate (50 μg / g), and Group E was treated with IMQ and intraperitoneal injection of 200 μl α-ketoglutarate (100 μg / g). The mice were treated three times a week for 10 weeks and then killed by CO2 and the indicators were measured. All mouse experiments were carried out in accordance with the animal ethics guidelines, and the animals were used in accordance with the guidelines of the Animal Ethics Committee of Jining Medical College (JNMC-2024-DW-101).

[0034] 2.2 SLE patient sample information The research protocol of the present invention has been approved by the Medical Ethics Committee of Jining Medical College (JNMC-2022-YX-006). All SLE patients and healthy subjects in the present invention provided written informed consent before the study. All patients were diagnosed according to the criteria revised by the American College of Rheumatology (1997), and the SLE Disease Activity Index (SLEDAI) was used to evaluate disease activity, and a critical value of ≥5 was used to define active disease. The SLE patients in the present invention do not include patients treated with high-dose immunocytotoxic therapy or steroids and patients with overlap syndrome, and peripheral blood samples are required to be collected from SLE patients and healthy donors. The inventors abided by the Helsinki Principles and obtained written and oral consent from each participant to ensure that the present invention complies with ethical guidelines.

[0035] 2.3 Isolation of human peripheral blood mononuclear cells (PBMCs) Blood samples from SLE patients and healthy participants were collected in heparinized tubes, and peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Ficoll-Paque™ PLUS.

[0036] 2.4 Isolation of mouse spleen B cells After grinding the mouse spleen, naive B cells were isolated using the Mouse B Cell Isolation Kit (BD); the purity of B cells isolated by negative selection was greater than 95%. The sorted B cells were then cultured in complete RPMI 1640 medium for subsequent operations.

[0037] 2.5 Real-time quantitative PCR analysis Total RNA was extracted using Trizol (purchased from Thermo Fisher Scientific) according to the manufacturer's instructions, and cDNA was prepared using the RevertAid First Strand cDNA Synthesis Kit (purchased from Thermo Fisher Scientific). Q-PCR analysis was performed using SYBR Green PCR Master Mix (purchased from Nanjing Novozyme Biotechnology Co., Ltd.) with GAPDH as the internal reference and 2 -ΔΔCt The relative gene expression was calculated using the formula, and all experiments were repeated three times.

[0038] 2.6 H&E staining The tissues were stably fixed in 4% paraformaldehyde solution for 24 h, then dehydrated through a series of gradient ethanol solutions and embedded in paraffin. The embedded tissue blocks were precisely cut into 4-μm ultrathin sections and stained with hematoxylin and eosin staining solution, observed under an optical microscope and semi-quantitatively blindly evaluated: Grade 0 indicated no proliferation; Grade 1 was that the proportion of proliferating cells in the total number of glomerular cells was less than 25%; Grade 2 was that 25% to 50% of the glomerular cells proliferated; and Grade 3 was that more than 50% of the glomerular cells proliferated, indicating a highly active inflammatory process.

[0039] 2.7 Immunofluorescence staining Kidney tissue sections were dewaxed and rehydrated using xylene and ethanol, and then nonspecific binding was blocked using 1% BSA. Subsequently, the tissue sections were incubated with Alexa Fluor 488-labeled goat anti-mouse IgG or IgM at 4°C overnight. The sections were washed with PBS containing 0.1% Tween-20 the next day and stained with DAPI. After staining, the tissue sections were placed under coverslips using Antifade mounting medium (Beyotime). Finally, the coverslips were sealed with an anti-fluorescence quencher, and the sections were observed under a fluorescence microscope (Olympus, Japan) and blindly evaluated for semiquantitative pathological changes. At the same time, we quantified the degree of IgG and IgM precipitation in the glomeruli: if there was no staining in the glomerulus, it was rated as grade 0; if the staining area was less than 25% of the total glomerular area, it was rated as grade 1 (weak); between 25% and 50% was grade 2 (moderate); and more than 50% was grade 3 (strong).

[0040] 2.8 Flow cytometry When performing cell phenotype staining analysis, cells are first collected and prepared into a homogenous single-cell suspension. Specific flow cytometry-specific antibodies are used for staining. When performing cell surface marker staining, cells are collected and prepared into a single-cell suspension, then stained with antibodies for specific phenotypes, incubated in the dark at 4°C for 30 min, washed with PBS, and then subjected to flow cytometry. Analyzed using the BD FACSVerse™ instrument, all FACS data were processed using Flowjo software. To ensure accurate results, isotype controls were included for each antibody used in the analysis.

[0041] 2.9 Enzyme-linked immunosorbent assay To measure the anti-ds-DNA level, we used a mouse anti-dsDNA IgG kit (purchased from Vibio Biotech Co., Ltd.) to analyze the MRL / lpr The levels of anti-dsDNA IgG in the sera of mice and IMQ mice were measured by absorbance at 450 nm using a microplate reader (BioTek microplate reader).

[0042] 2.10 Immunoblotting analysis Cells were collected and homogenized with lysis buffer and protein concentration was measured using a BCA protein assay kit (Beyotime). Proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a 0.45 μm polyvinylidene fluoride (PVDF) membrane. The membranes were blocked with 5% bovine serum albumin (BSA) for 2 h at room temperature and then incubated with primary antibodies at 4°C overnight. The next day, after washing five times, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (dilution 1:5000, Beyotime) at room temperature for 2 h. Protein expression was observed using an ECL kit (purchased from Thermo Fisher Scientific). β-actin was used as an internal control.

[0043] 2. Test results 1. The content of α-ketoglutarate in the serum of SLE patients and lupus model mice is significantly increased The contents of 100 organic acids in the serum of SLE patients and healthy controls were analyzed by organic acid targeted metabolomics. Figure 1 a) Compared with healthy controls, the levels of organic acids such as α-ketoglutaric acid, L-phenylalanine, and DL-pyroglutamic acid in the serum of SLE patients were significantly increased, while the levels of organic acids such as uracil, isophthalic acid, lactic acid, and itaconic acid were significantly decreased ( Figure 1b). In view of the fact that α-ketoglutarate is widely involved in the occurrence and development of various diseases, the present invention intends to focus on the effect of α-ketoglutarate on the pathogenesis of SLE. Therefore, the content of α-ketoglutarate in the serum of SLE patients and healthy controls was detected using clinical samples, and it was found that the content of α-ketoglutarate in the serum of SLE patients was indeed significantly higher than that of healthy controls ( Figure 1 c). It was also found that MRL / lpr The levels of α-ketoglutarate in the serum of spontaneous lupus model and IMQ-induced lupus model mice were significantly higher than those in control mice ( Figure 1 d and Figure 1 e).

[0044] The above results indicate that α-ketoglutarate is likely to be involved in the occurrence and development of SLE.

[0045] 2. α-ketoglutarate treatment significantly reduced MRL / lpr Lupus symptoms in mice In order to explore the regulatory effect of α-ketoglutarate on the pathogenesis of lupus, the present invention studied the effect of α-ketoglutarate on spontaneous lupus MRL / lpr The effect of the condition in mice was found to be MRL / lpr Compared with the blank control group, the MRL / lpr The degree of spleen enlargement in mice was significantly reduced ( Figure 2 a), spleen weight was significantly reduced ( Figure 2 b), the content of anti-double-stranded DNA (anti-dsDNA) antibodies in serum also decreased significantly ( Figure 2 c). H&E staining showed that α-ketoglutarate treatment could significantly reduce the MRL / lpr Renal injury in mice ( Figure 2 d). Moreover, immunofluorescence confocal microscopy revealed that MRL / lpr The deposition of IgG and IgM in the glomeruli of mice was significantly less than that of the control-treated MRL / lpr Mouse ( Figure 2 e).

[0046] The above results show that α-ketoglutarate can significantly alleviate the MRL / lpr The condition of lupus model mice.

[0047] 3. α-ketoglutarate treatment significantly inhibited MRL / lpr Activation and differentiation of immune cells in mice The present invention analyzes the effect of α-ketoglutarate on MRL / lpr The regulatory role of immune cell activation and differentiation in mice. lpr Compared with mice treated with α-ketoglutarate, MRL / lpr The expression of CD86 on the surface of B cells in the spleen and lymph nodes of mice was significantly reduced ( Figure 3 a. Figure 3 b. Figure 3 c and Figure 3 d), indicating that α-ketoglutarate significantly inhibited MRL / lpr Moreover, α-ketoglutarate treatment could significantly reduce MRL / lpr Germinal Center (GC) B cells in mouse spleen and lymph nodes ( Figure 3 e. Figure 3 f. Figure 3 g and Figure 3 h) and plasma cells ( Figure 3 i. Figure 3 j. Figure 3 k and Figure 3 l) ratio. This indicates that α-ketoglutarate can significantly inhibit the MRL / lpr Activation and differentiation of B cells in lupus mice.

[0048] The above results show that α-ketoglutarate can indeed inhibit MRL / lpr Activation and differentiation of B cells in mice.

[0049] 4. α-Ketoglutarate treatment significantly alleviated the symptoms of imiquimod-induced lupus mice In order to further study the regulatory effect of α-ketoglutarate on the occurrence and development of lupus, the present invention studied the effect of α-ketoglutarate on the condition of lupus model mice induced by imiquimod (IMQ) ( Figure 4 a) The results showed that the splenomegaly of IMQ mice treated with α-ketoglutarate was significantly reduced compared with that of IMQ mice treated with the control agent ( Figure 4 b), spleen weight was significantly reduced ( Figure 4 c), the content of anti-double-stranded DNA (anti-dsDNA) antibodies in serum also decreased significantly ( Figure 4 d). H&E staining showed that α-ketoglutarate treatment could significantly alleviate renal injury in IMQ mice ( Figure 4 e). Immunofluorescence confocal microscopy revealed that the deposition of IgG and IgM in the glomeruli of IMQ mice treated with α-ketoglutarate was significantly less than that of IMQ mice treated with the control agent ( Figure 4 f).

[0050] The above results indicate that α-ketoglutarate can significantly alleviate the condition of IMQ lupus model mice.

[0051] 5. α-Ketoglutarate treatment significantly inhibits the activation and differentiation of immune cells in imiquimod-induced lupus mice The present invention analyzes the regulatory effect of α-ketoglutarate on the activation and differentiation of immune cells in IMQ lupus mice. Figure 5 It was found that the expression of CD86 on the surface of B cells in the spleen of α-ketoglutarate-treated IMQ mice was significantly reduced compared with that of control-treated IMQ lupus mice ( Figure 5 a and Figure 5 b), indicating that α-ketoglutarate significantly inhibited the activation of B cells in IMQ mice. Moreover, α-ketoglutarate treatment significantly reduced the number of germinal center B cells in the spleen and lymph nodes of IMQ mice ( Figure 5 c and Figure 5 e) and plasma cells ( Figure 5 d and Figure 5 f) ratio.

[0052] The above results indicate that α-ketoglutarate can significantly inhibit the activation and differentiation of B cells in IMQ-induced lupus mice.

[0053] 6. In vitro experiments confirmed that α-ketoglutarate significantly inhibited the activation of TLRs signaling pathway in B cells Considering that TLR pathways, especially TLR4, TLR7 and TLR9 pathways, are crucial for the activation and differentiation of immune cells such as B cells, the present invention further studied whether α-ketoglutarate can regulate the activation of TLRs signaling pathways in immune cells such as B cells. Specifically, B220 cells sorted from the spleen of WT mice + B cells were treated with α-ketoglutarate (0 mM, 0.25 mM, 1 mM, 2.5 mM, 5 mM) for 24 h and then stimulated with lipopolysaccharide (100 ng / mL LPS), TLR7 agonist resiquimod (1 μg / mL R848), or stimulatory CpG1826 (1 μM CpG1826).

[0054] Consistent with the in vivo results, α-ketoglutarate pretreatment was found to significantly inhibit TLR ligand-mediated B cell surface CD86 ( Figure 6 a. Figure 6 c. Figure 6 e) and CD40 ( Figure 6 b. Figure 6 d. Figure 6 f) expression; Q-PCR results also showed that α-ketoglutarate could significantly inhibit TLR ligand-mediated TNF-α in B cells in a concentration-dependent manner ( Figure 6 g) and IL-12 ( Figure 6 h) expression; moreover, α-ketoglutarate significantly inhibited the phosphorylation of p65, p38, JNK and Erk proteins in R848-induced B cells ( Figure 6 i).

[0055] The above results suggest that α-ketoglutarate can indeed inhibit the activation of TLRs signaling pathway in B cells.

[0056] In summary, the present invention found that the content of α-ketoglutarate in the serum of SLE patients and lupus mice was abnormally increased, and α-ketoglutarate significantly inhibited TLR-mediated B cell activation and differentiation, thereby alleviating the occurrence of lupus.

[0057] Although preferred embodiments of the present invention have been described, additional changes and modifications may occur to these embodiments once those skilled in the art are aware of the basic inventive concepts.

[0058] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Application of α-ketoglutaric acid in the preparation of drugs for the treatment of systemic lupus erythematosus.

2. The use of α-ketoglutaric acid in the preparation of a drug for treating systemic lupus erythematosus according to claim 1, characterized in that: The alpha-ketoglutaric acid is used for preparing a medicine for alleviating spleen enlargement.

3. The use of α-ketoglutaric acid in the preparation of a drug for treating systemic lupus erythematosus according to claim 1, characterized in that: The alpha-ketoglutaric acid is used for preparing medicine for alleviating kidney damage.

4. The use of α-ketoglutaric acid in the preparation of a drug for treating systemic lupus erythematosus according to claim 1, characterized in that: The alpha-ketoglutaric acid is used for preparing a medicine for reducing the deposition of IgG and / or IgM in glomeruli.

5. The use of α-ketoglutaric acid in the preparation of a drug for treating systemic lupus erythematosus according to claim 1, characterized in that: The drug has α-ketoglutaric acid as the only active ingredient.

6. The use of α-ketoglutaric acid in the preparation of a drug for treating systemic lupus erythematosus according to claim 1, characterized in that: The drug also includes pharmaceutically acceptable excipients.

7. The use of α-ketoglutaric acid in the preparation of a drug for treating systemic lupus erythematosus according to claim 1, characterized in that: The medicine is an oral preparation or an injection.

8. The use according to claim 7, characterized in that: The oral preparation is any one of tablets, capsules, pills, powders, granules and syrups.

9. The use according to claim 7, characterized in that: The injection is injection solution or sterile powder for injection.

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