CN119745898B - Application of GppNHp in the preparation of drugs for treating systemic lupus erythematosus - Google Patents

Abstract

The present invention provides the application of GppNHp in the preparation of a drug for treating systemic lupus erythematosus, and belongs to the field of medical technology. The present invention can significantly alleviate various symptoms of lupus model mice 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 urine protein, reducing the concentration of tumor necrosis factor (TNF-α), and recovering 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.

Description

Application of GppNHp in preparing medicine for treating systemic lupus erythematosus

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to an application of GppNHp in preparing a medicine for treating systemic lupus erythematosus.

Background

Systemic Lupus Erythematosus (SLE) is a complex autoimmune disease, frequently seen in young women, whose pathological mechanisms are mainly involved in hyperactive B-cell and T-cell signaling, as well as abnormal release of cytokines. Currently, treatment of SLE relies primarily on widely used non-specific hormones and immunosuppressants. Although these treatments are able to control the condition and alleviate symptoms to some extent, long term use can lead to a range of side effects such as osteoporosis, increased risk of infection, kidney damage, etc.

The prior art treatments mainly include 1. Glucocorticoids, which are used to rapidly control inflammatory responses but cause serious side effects over prolonged use. 2. Antimalarial drugs (e.g. hydroxychloroquine) are used to treat light and moderate SLE and have immunomodulating effects but limited therapeutic efficacy. 3. Immunosuppressants (e.g., cyclophosphamide, etc.) are used to treat severe SLE, but may result in excessive suppression of the immune system and other serious side effects. 4. Biological agents (e.g., belieukinumab) are directed against specific immune cells or cytokines, are costly and may cause side effects such as infection, although they are efficacious. A common problem with these treatment regimens is that they cannot fundamentally modulate or correct immune abnormalities that lead to SLE, and are often accompanied by serious side effects, which limit their long-term use.

Existing treatments, while effective in controlling SLE symptoms, have significant shortcomings in terms of safety, persistence of efficacy, and cost effectiveness. In particular, long-term hormone and immunosuppressant dependent therapies may not only lead to a number of complications, but also gradually diminish the therapeutic efficacy for some patients. There is an urgent need to develop a new therapeutic approach to more safely and more effectively modulate the immune system of a patient, reduce the side effects of long-term treatment, and improve the quality of life of the patient. Programmed necrosis (Necroptosis) is a regulated form of cell death and has been found in recent years to be closely related to the pathogenesis of a variety of autoimmune diseases. Therefore, it is important to study a programmed necrosis inhibitor as an intervention drug for the treatment of SLE.

Disclosure of Invention

In view of the above, the invention aims to provide an application of GppNHp in preparing medicines for treating systemic lupus erythematosus. The invention significantly reduces various symptoms of lupus model mice, including reducing spleen and lymph node volume and weight, reducing kidney damage, reducing urine protein concentration, and modulating related immune inflammatory response indicators, through the use of a programmed necrosis inhibitor GppNHp. The invention provides a safe and effective preparation, which is expected to improve the life quality of patients.

In order to achieve the above object, the present invention provides the following technical solutions:

The invention provides an application of GppNHp in preparing a medicine for treating systemic lupus erythematosus.

Preferably, the GppNHp is used in a concentration of 5-15 mug/. Mu.L.

Preferably, the solvent of GppNHp is phosphate buffer.

Preferably, the GppNHp is used in the form of injection.

Preferably, the GppNHp is used for 1 to 5 days/time.

The invention also provides a medicament for treating systemic lupus erythematosus, which comprises GppNHp and a pharmaceutically acceptable carrier thereof.

Compared with the prior art, the invention has the following beneficial effects that the invention provides the application of GppNHp in preparing the medicine for treating the systemic lupus erythematosus. The invention can obviously relieve various symptoms of SLE by using the programmed necrosis inhibitor GppNHp, such as obviously reducing the volume and weight of spleen and lymph nodes, slowing down kidney injury, reducing the concentration of urine protein and inflammatory factors (tumor necrosis factor alpha (TNF-alpha)) and raising the concentration of complement C3. The preparation provided by the invention can effectively improve the symptom of lupus mice, and is hopeful to improve the life quality of SLE patients.

Drawings

FIG. 1 is a graph showing the results of the volume and weight of the spleen and axillary lymph nodes of mice before and after treatment with the inhibitor GppNHp, wherein A is a graph showing the results of the spleen volume of mice before and after treatment with the inhibitor GppNHp, B is a graph showing the results of the spleen weight of mice before and after treatment with the inhibitor GppNHp, C is a graph showing the results of the axillary lymph node volume of mice before and after treatment with the inhibitor GppNHp, and D is a graph showing the results of the axillary lymph node weight of mice before and after treatment with the inhibitor GppNHp;

FIG. 2 is a graph of HE staining of kidney pathology in mice before and after treatment with a therapeutic inhibitor GppNHp of apoptosis, on a scale of 20 μm;

FIG. 3 is a graph showing changes in levels of autoantibodies (anti-dsDNA antibodies), complement C3, and TNF- α levels of the therapeutic agents GppNHp;

FIG. 4 is a graph showing changes in serum creatinine and urine protein concentration before and after treatment with a therapeutic agent GppNHp for apoptosis;

FIG. 5 is a graph showing the viability of peripheral blood neutrophils in mice before and after flow cytometry detection treatment;

In fig. 1 to 5, MRL/Mpj represents a normal mouse group, MRL/lpr+dmso represents a non-treated lupus model mouse group, DMSO alone was used as a control, MRL/lpr+ GppNHp represents a lupus model mouse group treated with a programmed necrosis inhibitor GppNHp, and MRL/lpr+ctx represents a lupus model mouse group treated with a clinical first-line drug Cyclophosphamide (CTX).

Detailed Description

The invention provides an application of GppNHp in preparing a medicine for treating systemic lupus erythematosus.

According to the invention, gppNHp is dissolved in phosphate buffer solution, sterile filtration is carried out to obtain GppNHp injection with the concentration of 5-15 mu g/mu L, when GppNHp injection is used for mice, injection is carried out according to the use amount of 0.5-1.5 mu L/g weight of the mice, the use frequency of 1-5 days/time is used for 8-15 times continuously, the concentration of GppNHp injection is preferably 8-12 mu g/mu L, more preferably 10 mu g/mu L, the concentration of phosphate buffer solution is 0.001-0.02M, preferably 0.005-0.015M, more preferably 0.01M, the pH of phosphate buffer solution is 7.0-7.6, preferably 7.1-7.5, more preferably 7.2-7.4, the use amount of GppNHp is preferably 0.8-1.2 mu L/g weight of mice, more preferably 1-4 days/g weight of mice, more preferably 2-3 days/time is further preferably used for 3 days continuously.

The invention also provides a medicament for treating systemic lupus erythematosus, which comprises GppNHp and a pharmaceutically acceptable carrier thereof.

In the present invention, the pharmaceutical agent is preferably an injection comprising GppNHp.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Examples

1. Experimental materials and preparation

1) Experimental animals Male MRL/lpr mice (purchased from Sai Biotechnology Co., ltd.) of 14-16 weeks old are selected and used widely in disease models because of their autoimmune properties similar to human systemic lupus erythematosus.

2) Preparation of the drug GppNHp (available from MCE company under the trade designation HY-137167) was dissolved in phosphate buffer (PBS, 0.01M,pH 7.2~7.4) at a concentration of 10. Mu.g/. Mu.L. All solutions were sterile filtered through a 0.22 μm filter before use.

2. Design of experiment

1) Grouping and treatment MRL/lpr mice were randomized into four groups, with treatment groups being intraperitoneally injected with a solution of a programmed necrosis inhibitor GppNHp (MRL/lpr+ GppNHp), and control groups being injected with equal volumes of DMSO (MRL/lpr+DMSO) and CTX (MRL/lpr+CTX, CTX available from Sigma). The injection was performed every 3 days at a dose of 1. Mu.L/g body weight for 10 consecutive times. Male MRL/Mpj mice (purchased from Sai Biotechnology Co., ltd.) of 14-16 weeks old were selected as healthy control group without any treatment.

2) Physiological index monitoring the mice were monitored daily for body weight, food and water intake during injection to assess potential toxicity of the drug and effect on overall animal health.

3. Collection and analysis of biological samples

1) Urine and blood samples urine and venous blood from mice were collected the third day after the last injection of the experiment. Urine was stored at-80 ℃ for subsequent urine protein concentration analysis. Blood was added to heparin-containing tubes, left to stand at 25℃for 30min, centrifuged at 3000rpm for 15min at 4℃and the supernatant collected for biochemical analysis.

2) Tissue samples, namely, after blood is taken from eyeballs, mice are killed, spleen, lymph nodes, kidneys and other related organs are collected, and weight and volume measurements are carried out, and the results are shown in figure 1. Each organ was fixed in 4% paraformaldehyde and then HE stained to assess pathological changes in the tissue, the results are shown in figure 2.

4. Biomarker analysis

1) Immunoassay the levels of TNF-alpha, complement C3, and anti-double stranded DNA (dsDNA) antibodies in serum were determined using ELISA kits and the results are shown in FIG. 3.

Renal function testing serum creatinine and urine protein concentrations were measured and the extent of kidney injury was assessed and the results are shown in figure 4.

2) Flow cytometry analysis peripheral blood neutrophils were isolated using a Miltenyi Biotec kit (available from Meinaand Biotechnology Co., germany) and the viability of the cells was measured and the results are shown in FIG. 5.

5. Data processing and statistical analysis

Statistical method statistical analysis was performed using Graphpad prism8.0 software. Metering data to The 2 sets of comparisons are shown using the t-test. P <0.05 indicates a statistical difference between the two.

6. Experimental results and assessment

As can be seen from fig. 1, the mice after treatment with GppNHp had significantly smaller spleen and axillary lymph nodes and significantly lighter weight compared to the MRL/lpr+dmso control group, demonstrating that the use of GppNHp significantly reduced spleen and axillary lymph nodes volume and weight. As can be seen from FIG. 2, the normal mouse group (MRL/Mpj) represents a healthy control, the glomeruli are relatively intact in structure and closely packed in cells, and no obvious pathological features are seen. The classical lupus model mice group (MRL/lpr+DMSO) is not treated, and only DMSO is used as a control, so that the glomerulus structure is obviously abnormal, the glomerulus basement membrane is thickened, the cell arrangement is disordered, and inflammatory cell infiltration is accompanied, so that serious pathological damage exists in the local part of the kidney. Mice treated with the apoptosis inhibitor GppNHp (MRL/lpr+ GppNHp) had improved glomerular structure, reduced basement membrane thickening, tighter cell arrangement and reduced inflammatory infiltration compared to untreated MRL/lpr+DMSO groups, indicating that GppNHp treatment has some improvement in renal pathological injury. CTX (cyclophosphamide) is a commonly used immunosuppressant currently in clinical treatment of SLE patients. Using CTX-treated mice (MRL/lpr+ctx) as a positive control, the pathological lesions of the glomeruli were significantly reduced compared to the MRL/lpr+dmso group, with an improvement in tissue architecture approaching normal, but with a small inflammatory cell infiltration. As can be seen from fig. 3-5, urine protein concentration and blood creatinine concentration decreased significantly after GppNHp treatment compared to the MRL/lpr+dmso control group, indicating that GppNHp was able to slow down kidney injury in lupus mice, and complement C3 concentration increased after GppNHp treatment with anti-dsDNA antibodies, tumor necrosis factor TNF- α concentration decreased. Therefore, the programmed necrosis inhibitor GppNHp can correct abnormal autoimmune reaction in the lupus mice by regulating the immunocyte activity, thereby remarkably relieving the lupus phenotype of the mice, providing important theoretical and practical basis for developing a new strategy for treating systemic lupus erythematosus, and having wide application prospect.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (4)

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 GppNHp is 5-15 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.