Application of FosB siRNA in the preparation of drugs for treating IgA nephropathy

Abstract

This invention discloses the application of FosB siRNA in the preparation of drugs for treating IgA nephropathy, which can significantly reduce the degree of renal mesangial proliferation and alleviate renal pathological changes, and has the advantages of high efficiency, safety, and ease of production. The mechanism of action of FosB siRNA is: inhibiting mesangial cell proliferation and alleviating renal pathological changes.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more particularly to the application of FosB siRNA in the preparation of drugs for treating IgA nephropathy. Background Technology

[0002] IgA nephropathy (IgAN) remains the most common primary glomerulonephritis worldwide and the leading cause of end-stage renal disease (ESRD). Approximately 20-40% of patients will slowly but steadily progress to ESRD within 10-20 years after onset. It is estimated that IgA nephropathy accounts for up to 10% of all ESRD patients requiring renal replacement therapy in Western countries. Related multicenter studies have also found that the 10-year relapse rate of this disease is as high as 42% in adult patients who have undergone kidney transplantation. Many patients progress to ESRD at a relatively young age, placing a heavy burden on their personal health and socioeconomic status. Treatment for IgA nephropathy has not changed significantly to date. Guideline-recommended treatment still focuses on traditional non-immunosuppressive therapies, such as blood pressure control, lifestyle modifications, and appropriate suppression of the renin-angiotensin system (RAS). However, even with adequate RAS suppression and optimal blood pressure control, patients with IgA nephropathy still face a significant risk of renal function progression.

[0003] The pathogenesis of IgA nephropathy is not fully understood, but the "multiple hit" theory is currently the accepted approach. Recent treatments for IgA nephropathy have included traditional and novel corticosteroids, as well as traditional immunosuppressants and complement inhibitors. While these treatments have shown some efficacy, traditional corticosteroids can cause side effects such as increased risk of infection, cataracts, osteoporosis, fractures, and coronary artery disease. Even with novel corticosteroids (such as budesonide released in the gut), the safety and efficacy require further clinical validation. Traditional immunosuppressants, such as cyclophosphamide, are associated with increased risk of premature ovarian failure, hemorrhagic cystitis, bladder and other malignancies, leukopenia, and increased risk of infection. Complement inhibitors for the treatment of IgA nephropathy, such as the C3 inhibitor APL-2, the C5a receptor antagonist avacopan (also known as CCX168), the selective oral factor B inhibitor LNP023, the monoclonal antibody OSM721 (MBL-associated serine protease 2, MASP-2), and inhibitors of C5b-9, are currently still in phase II or III clinical trials, and their efficacy and related risks remain unclear.

[0004] Glomerular mesangial cell proliferation and increased extracellular matrix are important pathological features of IgA nephropathy and significant factors influencing disease progression and prognosis. Mesangial cells and their secreted extracellular matrix constitute the supporting framework of glomerular vascular loops, playing a crucial role in maintaining glomerular structural and functional homeostasis and enabling glomerular responses to damage. Patients with IgA nephropathy show a significant increase in local angiotensin II and marked proliferation of glomerular mesangial cells. Despite the widespread use of RAS inhibitors, some patients still face the risk of disease progression. Currently, there are no drugs that can specifically inhibit glomerular mesangial cell proliferation. Therefore, exploring the specific pathogenesis of IgA nephropathy and developing targeted drugs to delay the progression of renal function in patients are urgent problems that the scientific community needs to solve. Summary of the Invention

[0005] To overcome the shortcomings of the prior art, the technical problem to be solved by the present invention is to provide a FosB siRNA for use in the preparation of drugs for treating IgA nephropathy, which can significantly reduce the degree of renal pathological mesangial proliferation and alleviate renal pathological changes, and has the advantages of high efficiency, safety and easy production.

[0006] The technical solution of the present invention is: the application of FosB siRNA in the preparation of drugs for treating IgA nephropathy, wherein FosB siRNA inhibits mesangial cell proliferation and alleviates pathological changes in the kidney.

[0007] Preferably, FosB siRNA reduces C5aR expression.

[0008] The method for using FosB siRNA in the preparation of drugs for treating IgA nephropathy is also provided, including the following steps:

[0009] (1) The human glomerular mesangial cell line was resuspended in DMEM low-glucose medium containing 2% fetal bovine serum, and the initial density was adjusted to 1×10⁶ cells / mL. 6 / mL;

[0010] (2) Transfect mesangial cells with FosB siRNA at a concentration of 50 nM for 24-48 hours, and then stimulate them with culture medium containing 5% IgA nephropathy patient serum for 24 hours.

[0011] (3) The control group was given the same dose and the same time of PBS, and then stimulated with culture medium containing serum from IgA nephropathy patients for 24 hours after the same time.

[0012] (4) Cell proliferation was detected by CCK-8 assay, and changes in the content of extracellular matrix in cell supernatant were detected by enzyme-linked immunosorbent assay.

[0013] Preferably, the method further includes step (5), obtaining a kidney tissue specimen from a patient, extracting and culturing primary glomerular mesangial cells, transfecting the cells according to steps (1)-(2), and detecting cell proliferation and extracellular matrix content according to step (4).

[0014] This invention provides a novel drug for treating IgA nephropathy, namely, containing an effective dose of FosB siRNA and pharmaceutical excipients.

[0015] This drug can significantly reduce the degree of renal pathological mesangial proliferation and alleviate renal pathological changes, thus effectively treating IgA nephropathy. Moreover, as an RNA drug, it has the characteristics of high efficiency, safety, and ease of production. Attached Figure Description

[0016] Figure 1 The image shows the pathological features of renal mesangial proliferation in IgA nephropathy model mice and after treatment with small interfering RNA (PAS staining, ×400; left image is the model group, with the black arrow indicating significant mesangial proliferation; right image is the treatment group).

[0017] Figure 2 The results show the comparison of glomerular mesangial cell proliferation in different treatment groups as detected by CCK-8 assay and the content of fibronectin (FN), laminin (LN), and type IV collagen (ColIV) in the cell supernatant as detected by ELISA. *P<0.05.

[0018] Figure 3 The results of Western blot show that inhibiting FosB significantly reduces C5aR expression.

[0019] Figure 4 The study showed a comparison of the proliferation of primary glomerular mesangial cells in human kidney tissues under different treatment groups (CCK-8) and changes in the content of extracellular matrix secreted in the supernatant (ELISA), *P<0.05. Detailed Implementation

[0020] This invention provides a novel drug for treating IgA nephropathy, comprising an effective dose of FosB siRNA and a pharmaceutical excipient. The mechanism of action of FosB siRNA is to inhibit mesangial cell proliferation and alleviate renal pathological changes. Furthermore, FosB siRNA reduces C5aR expression.

[0021] The embodiments of the present invention will be described in detail below.

[0022] The FosB siRNA treatment regimen is as follows:

[0023] 1. Treatment of animal models: Animal models (12-week-old induced model mice) were treated with FosB siRNA (purchased from Guangzhou Ruibo Biotechnology Co., Ltd., catalog number siBDMV002) via tail vein injection, with a total dose of 400ug per mouse, twice a week for 7 consecutive weeks; the control group was treated with physiological saline at the same dose and frequency.

[0024] 2. Cell therapy: Human glomerular mesangial cell lines were resuspended in DMEM low-glucose medium containing 2% fetal bovine serum, and the initial density was adjusted to 1×10⁻⁶ cells / day. 6 / mL. FosB siRNA (purchased from Guangzhou Ruibo Biotechnology Co., Ltd., catalog number stB0003607C-1-5) was transfected into mesangial cells at a concentration of 50 nM for 24-48 hours, followed by stimulation with culture medium containing 5% IgA nephropathy patient serum for 24 hours. The control group received the same dose and duration of PBS, followed by stimulation with IgA nephropathy patient serum for 24 hours. Cell proliferation was assessed using CCK-8 assay, and changes in extracellular matrix content in the cell supernatant were detected using enzyme-linked immunosorbent assay (ELISA). Simultaneously, kidney tissue samples were obtained from patients, primary glomerular mesangial cells were extracted and cultured, and transfected using the above method. Cells were then stimulated with culture medium containing 5% IgA nephropathy patient serum for 24 hours, and cell proliferation and extracellular matrix content were assessed.

[0025] This invention is the first to discover that FosB siRNA can be used as a novel drug for treating IgA nephropathy. Animal experiments showed that mice induced with IgA nephropathy were treated with saline via tail vein injection and with animal-derived FosB siRNA. Compared with the control group, the treatment group showed reduced mesangial proliferation. Mesangial cell proliferation is an important pathological feature of IgA nephropathy, and inhibiting mesangial cell proliferation and alleviating pathological changes in the kidneys is one of the therapeutic targets. Cellular experiments on the treatment of human glomerular mesangial cells with FosB siRNA showed that FosB siRNA can inhibit the proliferation of glomerular mesangial cells and reduce extracellular matrix secretion, suggesting that FosB siRNA achieves its therapeutic effect on IgA nephropathy by inhibiting mesangial cell proliferation.

[0026] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0027] Example 1: Animal experiment on FosB siRNA treatment of IgA nephropathy

[0028] Establishment of IgA nephropathy mouse model: The mouse model of IgA nephropathy was induced by a combination of gavage with low endotoxin bovine serum albumin and tail vein injection of staphylococcal enterotoxin B. The specific induction process can be found in the literature (Wan Q, Zhou J, Wu Y, et al. Ren Fail, 2022, 44(1): 1216-1226.). After one week of acclimatization, the model group mice were given 0.5 ml of acidified water with 0.1% bovine serum albumin (BSA, catalog number 9048-46-8, purchased from Yeasen Science and Technology Co., Ltd.) by gavage every other day. At the same time, starting from week 6, 0.2 ml of 2% BSA buffer was injected intravenously once a day for 3 consecutive days. BSA gavage was stopped in week 9, and 0.2 ml of 0.1 mg / ml staphylococcal enterotoxin B (SEB, Academy of Military Medical Sciences, Beijing, China) buffer was injected into the tail vein once a week for 3 consecutive weeks. The entire modeling process lasted 12 weeks. The control group mice were also given saline at the same dose, administration method and frequency for 12 weeks.

[0029] Treatment of the animal model: FosB siRNA and control saline were administered via tail vein injection to 12-week-old IgA nephropathy-induced model mice. The total dose per mouse was 400 μg, administered twice weekly for 7 weeks. The control group received the same dose and frequency of saline. Periodic acid-Schiff (PAS) staining was used to examine the degree of mesangial proliferation in the renal tissue. Compared with the saline control group, the degree of mesangial proliferation in the FosB siRNA treatment group was lower. Specific results are detailed below. Figure 1 This indicates that the drug has a certain effect on inhibiting the proliferation of renal mesangial cells in IgA nephropathy. No adverse events such as malignant tumors or infections occurred in the mice treated with the above methods.

[0030] Example 2 Cell Experiment

[0031] To further verify the broad-spectrum significance of FosB siRNA in the treatment of IgA nephropathy and the mechanism by which this drug inhibits the proliferation of mesangial cells in IgA nephropathy, we transfected human glomerular mesangial cells with FosB siRNA.

[0032] The target cells (human glomerular mesangial cells) were resuspended in DMEM low-glucose medium containing 2% fetal bovine serum, and the density was adjusted to 1×10⁶ cells / day. 6 / ml. FosB siRNA was transfected at a concentration of 50 nM for 24-48 hours, followed by stimulation with culture medium containing 5% serum from IgA nephropathy patients for 24 hours. The control group received the same dose and duration of PBS, followed by stimulation with culture medium containing 5% serum from IgA nephropathy patients for 24 hours. Complement C5a / C5aR activation is an important mechanism in the pathogenesis and progression of IgA nephropathy and a significant factor leading to mesangial cell proliferation. It is hypothesized that FosB siRNA can inhibit glomerular mesangial cell proliferation and reduce extracellular matrix secretion by reducing C5aR expression. Western blot results after treatment showed that FosB siRNA could reduce C5aR expression (C5aR antibody purchased from Proteintech Group, Inc., catalog number 21316-1-AP), alleviate mesangial cell proliferation and extracellular matrix secretion, suggesting the significance of FosB siRNA in treating IgA nephropathy by reducing C5aR expression. See details below. Figure 2-3 .

[0033] To further verify that the significance of FosB siRNA in treating IgA nephropathy is not limited to animal models and cell lines, kidney tissue specimens were obtained from patients. Primary glomerular mesangial cells were extracted according to the method described in the literature (Yin Yanzhi, Zhang Limei, Ju Jianwei, et al. Chinese Journal of Tissue Engineering Research and Clinical Rehabilitation, 2010, 14(11):1939-1942.) and cultured. After transfection for 24-48 hours, the cells were stimulated with a culture medium containing 5% serum from IgA nephropathy patients. The results showed that mesangial cell proliferation was inhibited and extracellular matrix secretion was reduced. For details, see [link to relevant documentation]. Figure 4 .

[0034] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.

Claims

1. The application of FosB siRNA in the preparation of drugs for treating IgA nephropathy, characterized by: The FosB siRNA is from Guangzhou Ruibo Biotechnology Co., Ltd., with product numbers stB0003607C-1-5 or siBDMV002. The FosB siRNA inhibits mesangial cell proliferation and alleviates pathological changes in the kidney.

2. The application of FosB siRNA according to claim 1 in the preparation of drugs for treating IgA nephropathy, characterized in that: FosB siRNA reduces C5aR expression.

3. The use of FosB siRNA according to claim 1 or 2 in the preparation of drugs for treating IgA nephropathy, characterized in that: Drugs for treating IgA nephropathy contain an effective dose of FosB siRNA and pharmaceutical excipients.

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