Application of GppNHp in preparation of medicine for treating systemic lupus erythematosus

Abstract

The invention provides application of GppNHp in preparation of a medicine for treating systemic lupus erythematosus, and belongs to the technical field of medicines. By using the programmed necrosis inhibitor GppNHp, various symptoms of lupus model mice can be remarkably relieved, for example, the volume and weight of the spleen and lymph nodes are remarkably reduced, kidney injury is slowed down, urine protein is reduced, the concentration of a tumor necrosis factor (TNF-alpha) is reduced, and the concentration of complement C3 is recovered. The preparation provided by the invention can effectively improve the symptoms of lupus mice, and is expected to improve the life quality of SLE patients.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to the application of GppNHp in the preparation of drugs for treating systemic lupus erythematosus. Background Art

[0002] Systemic lupus erythematosus (SLE) is a complex autoimmune disease that occurs frequently in young women. Its pathological mechanism mainly involves overactive B-cell and T-cell signaling, as well as abnormal release of cytokines. Currently, the treatment of SLE mainly relies on widely used nonspecific hormones and immunosuppressants. Although these treatments can control the disease and relieve symptoms to a certain extent, long-term use will bring a series of side effects, such as osteoporosis, increased risk of infection, and kidney damage.

[0003] The treatment methods in the prior art mainly include: 1. Glucocorticoids: used to quickly control inflammatory responses, but long-term use can cause serious side effects. 2. Antimalarial drugs (such as hydroxychloroquine): used to treat mild to moderate SLE, have immunomodulatory effects, but the therapeutic effect is limited. 3. Immunosuppressants (such as cyclophosphamide, etc.): used to treat severe SLE, but may lead to excessive suppression of the immune system and other serious side effects. 4. Biological agents (such as belimumab): targeting specific immune cells or cytokines, although the efficacy is significant, but the cost is high and may cause side effects such as infection. The common problem of these treatment options is that they cannot fundamentally regulate or correct the immune abnormalities that cause SLE, and are often accompanied by serious side effects, which limits their long-term use.

[0004] Although existing treatments have played a role in controlling SLE symptoms, they still have significant deficiencies in terms of safety, sustainability of efficacy, and cost-effectiveness. In particular, long-term reliance on hormone and immunosuppressant treatment may not only lead to a variety of complications, but also gradually weaken the therapeutic effect for some patients. There is an urgent need to develop a new treatment method to regulate the patient's immune system more safely and effectively, reduce the side effects of long-term treatment, and improve the patient's quality of life. Programmed necrosis is a regulated form of cell death that has been found to be closely related to the pathogenesis of a variety of autoimmune diseases in recent years. Therefore, it is particularly important to study a programmed necrosis inhibitor as an intervention drug for the treatment of SLE. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide the use of GppNHp in the preparation of a drug for treating systemic lupus erythematosus. The present invention significantly alleviates various symptoms of lupus model mice by using the programmed necrosis inhibitor GppNHp, including reducing the volume and weight of the spleen and lymph nodes, slowing down kidney damage, reducing urine protein concentration, and regulating related immune inflammatory response indicators. The present invention provides a safe and effective preparation that is expected to improve the quality of life of patients.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides application of GppNHp in preparing a medicine for treating systemic lupus erythematosus.

[0008] Preferably, the concentration of GppNHp is 5 to 15 μg / μL.

[0009] Preferably, the solvent of the GppNHp is phosphate buffer.

[0010] Preferably, the GppNHp is administered by injection.

[0011] Preferably, the GppNHp is used 1 to 5 times per day.

[0012] The present invention also provides a medicament for treating systemic lupus erythematosus, comprising GppNHp and a pharmaceutically acceptable carrier thereof.

[0013] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides the use of GppNHp in the preparation of a drug for treating systemic lupus erythematosus. The present invention can significantly alleviate various symptoms of SLE by using the programmed necrosis inhibitor GppNHp, such as significantly reducing the volume and weight of the spleen and lymph nodes, slowing down kidney damage, reducing the concentration of urine protein and inflammatory factors (tumor necrosis factor α (TNF-α)), and restoring the concentration of complement C3. The preparation provided by the present invention can effectively improve the symptoms of lupus mice, and is expected to improve the quality of life of SLE patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The figures are the volume and weight results of the spleen and axillary lymph nodes of mice before and after the treatment with the programmed necrosis inhibitor GppNHp, wherein A is the volume of the spleen of mice before and after the treatment with the programmed necrosis inhibitor GppNHp, B is the weight of the spleen of mice before and after the treatment with the programmed necrosis inhibitor GppNHp, C is the volume of the axillary lymph nodes of mice before and after the treatment with the programmed necrosis inhibitor GppNHp, and D is the weight of the axillary lymph nodes of mice before and after the treatment with the programmed necrosis inhibitor GppNHp;

[0015] Figure 2 HE staining of mouse kidney pathology before and after treatment with programmed necrosis inhibitor GppNHp, scale bar is 20μm;

[0016] Figure 3 The graphs show the changes in the levels of autoantibodies (anti-dsDNA antibodies), complement C3, and tumor necrosis factor TNF-α before and after treatment with the programmed necrosis inhibitor GppNHp;

[0017] Figure 4 This is a graph showing changes in serum creatinine and urine protein concentrations before and after treatment with the programmed necrosis inhibitor GppNHp;

[0018] Figure 5 This is a graph showing the viability of neutrophils in the peripheral blood of mice before and after treatment detected by flow cytometry;

[0019] Figure 1 to Figure 5 Here, MRL / Mpj represents the normal mouse group, MRL / lpr+DMSO represents the lupus model mouse group that did not receive treatment, with only DMSO used as a control, MRL / lpr+GppNHp represents the lupus model mouse group treated with the programmed necrosis inhibitor GppNHp, and MRL / lpr+CTX represents the lupus model mouse group treated with the clinical first-line drug cyclophosphamide (CTX). DETAILED DESCRIPTION

[0020] The present invention provides application of GppNHp in preparing medicine for treating systemic lupus erythematosus.

[0021] In the present invention, GppNHp is dissolved in a phosphate buffer solution and sterile filtered to obtain a GppNHp injection solution with a concentration of 5 to 15 μg / μL. When the GppNHp injection solution is used for mice, it is injected at a dosage of 0.5 to 1.5 μL / g of mouse body weight and a frequency of 1 to 5 days / time, and is used continuously for 8 to 15 times; the concentration of the GppNHp injection solution is preferably 8 to 12 μg / μL, more preferably 10 μg / μL, and the concentration of the phosphate buffer solution is 0.001 to 0.02 M, preferably 0.0 05~0.015M, more preferably 0.01M, the pH of the phosphate buffer is 7.0~7.6, preferably 7.1~7.5, more preferably 7.2~7.4, the sterile filtration uses a filter membrane with a pore size of 0.22μm, the amount of GppNHp used is preferably 0.8~1.2μL / g mouse body weight, more preferably 1μL / g mouse body weight, the frequency of use is preferably 2~4 days / time, more preferably 3 days / time, the number of consecutive uses is preferably 9~12 times, more preferably 10 times in succession.

[0022] The present invention also provides a medicament for treating systemic lupus erythematosus, comprising GppNHp and a pharmaceutically acceptable carrier thereof.

[0023] In the present invention, the medicament is preferably an injection containing GppNHp.

[0024] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0025] Example

[0026] 1. Experimental Materials and Preparation

[0027] 1) Experimental animals: Male MRL / lpr mice aged 14 to 16 weeks (purchased from Saiye Biotechnology Co., Ltd.) were selected. This type of mouse has autoimmune characteristics similar to those of human systemic lupus erythematosus and is widely used as a disease model.

[0028] 2) Drug preparation: GppNHp (purchased from MCE, catalog number HY-137167) was dissolved in phosphate buffered saline (PBS, 0.01 M, pH 7.2-7.4) at a concentration of 10 μg / μL. All solutions were sterile filtered through a 0.22 μm filter membrane before use.

[0029] 2. Experimental Design

[0030] 1) Grouping and treatment: MRL / lpr mice were randomly divided into four groups. The treatment group was intraperitoneally injected with the programmed necrosis inhibitor GppNHp solution (MRL / lpr+GppNHp), and the control group was injected with equal volumes of DMSO (MRL / lpr+DMSO) and CTX (MRL / lpr+CTX, CTX purchased from Sigma). The injection was once every 3 days, with each injection dose of 1 μL / g body weight, for 10 consecutive times. Male MRL / Mpj mice aged 14 to 16 weeks (purchased from Saiye Biotechnology Co., Ltd.) were selected as the healthy control group without any treatment.

[0031] 2) Physiological indicator monitoring: During the injection process, the body weight, food and water intake of mice were monitored daily to assess the potential toxicity of the drug and its impact on the overall health of the animals.

[0032] 3. Collection and Analysis of Biological Samples

[0033] 1) Urine and blood samples: On the third day after the last injection of the experiment, urine and venous blood were collected from mice. Urine was stored at -80°C for subsequent urine protein concentration analysis. Blood was added to a tube containing heparin, allowed to stand at 25°C for 30 minutes, and then centrifuged at 3000 rpm at 4°C for 15 minutes, and the supernatant was collected for biochemical analysis.

[0034] 2) Tissue samples: After removing the eyeballs and drawing blood, the mice were killed and the spleen, lymph nodes, kidneys and other related organs were collected for weight and volume measurement. The results are shown in Figure 1 Each organ was fixed in 4% paraformaldehyde and then stained with HE to evaluate the pathological changes of the tissue. The results are shown in Figure 2 .

[0035] 4. Biomarker Analysis

[0036] 1) Immunoassay: ELISA kits were used to measure the levels of TNF-α, complement C3 and anti-double-stranded DNA (dsDNA) antibodies in serum. Figure 3 .

[0037] Renal function test: Serum creatinine and urine protein concentrations are measured to assess the extent of kidney damage. Results are shown in Figure 4 .

[0038] 2) Flow cytometric analysis: Peripheral blood neutrophils were isolated using a Miltenyi Biotec kit (purchased from Miltenyi Biotec GmbH, Germany) and the cell viability was tested. The results are shown in Figure 5 .

[0039] 5. Data Processing and Statistical Analysis

[0040] Statistical methods: Graphpad Prism8.0 software was used for statistical analysis. The t test was used to compare the two groups. P < 0.05 indicated that there was a statistically significant difference between the two groups.

[0041] 6. Experimental Results and Evaluation

[0042] Depend on Figure 1 It can be seen that compared with the MRL / lpr+DMSO control group, after treatment with GppNHp, the volume of the spleen and axillary lymph nodes of mice was significantly reduced and the weight was significantly lighter, indicating that the use of GppNHp can significantly reduce the volume and weight of the spleen and axillary lymph nodes. Figure 2It can be seen that the normal mouse group (MRL / Mpj) represents a healthy control: the structure of the glomerulus is relatively complete, the cells are closely arranged, and no obvious pathological features are observed. The classic lupus model mouse group (MRL / lpr+DMSO) did not receive treatment and only used DMSO as a control: the structure of the glomerulus was obviously abnormal, with thickening of the glomerular basement membrane, disordered cell arrangement, and inflammatory cell infiltration, indicating that there is severe pathological damage to the local kidney. Compared with the untreated MRL / lpr+DMSO group, the group of mice treated with the programmed necrosis inhibitor GppNHp (MRL / lpr+GppNHp) showed improved glomerular structure, alleviated basement membrane thickening, more closely arranged cells, and reduced inflammatory infiltration, indicating that GppNHp treatment has a certain improvement effect on renal pathological damage. CTX (cyclophosphamide) is a commonly used immunosuppressant for the clinical treatment of SLE patients. The CTX-treated mice group (MRL / lpr+CTX) was used as a positive control. Compared with the MRL / lpr+DMSO group, the pathological damage of the glomeruli was significantly reduced, and the tissue structure improved to near normal, but there was still a small amount of inflammatory cell infiltration. Figure 3 to Figure 5 It can be seen that compared with the MRL / lpr+DMSO control group, after treatment with GppNHp, the urine protein concentration and blood creatinine concentration decreased significantly, indicating that GppNHp can slow down kidney damage in lupus mice; after treatment with GppNHp, the concentrations of anti-dsDNA antibodies and tumor necrosis factor TNF-α decreased, and the concentration of complement C3 increased. It can be seen that the programmed necrosis inhibitor GppNHp can correct the abnormal autoimmune response in lupus mice by regulating the activity of immune cells, thereby significantly alleviating the lupus phenotype of mice, which provides an important theoretical and practical basis for the development of new strategies for the treatment of systemic lupus erythematosus and has broad application prospects.

[0043] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of GppNHp in the preparation of drugs for the treatment of systemic lupus erythematosus.

2. The use according to claim 1, characterized in that: The concentration of the GppNHp is 5 to 15 μg / μL.

3. The use according to claim 2, characterized in that: The solvent of the GppNHp is phosphate buffer.

4. The use according to claim 3, characterized in that: The GppNHp is used by injection.

5. The use according to claim 4, characterized in that: The GppNHp is used 1 to 5 times a day.

6. A medicament for treating systemic lupus erythematosus, characterized in that: The medicament comprises GppNHp and a pharmaceutically acceptable carrier thereof.

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