Use of an e3 ubiquitin ligase siah2 inhibitor in the preparation of a drug for protecting the kidney from lupus nephritis

Abstract

The application discloses application of an E3 ubiquitin ligase SIAH2 inhibitor in preparation of a lupus nephritis kidney protection drug. Researches of the application show that the SIAH2-LATS2 axis participates in the pathogenic process of kidney fibrosis of lupus nephritis, and it is confirmed that LATS2 can be used as a target point for preventing fibrosis progression in lupus nephritis. It is shown that LATS2 is abnormally down-regulated in lupus nephritis, and the use of an E3 ubiquitin ligase SIAH2 inhibitor can restore the LATS2 level, reduce the production of a LATS2 downstream gene CTGF, reduce the occurrence of interstitial fibrosis, reduce the proteinuria effect of lupus nephritis, thereby protecting the kidney function and delaying the progression of lupus nephritis. The application uses the SIAH2 inhibitor vitamin K3, which can prevent the conversion of lupus nephritis to ESRD, and can be used as an effective drug for treating and preventing kidney fibrosis, and provides a new selection for lupus nephritis treatment drugs and fibrosis prevention and treatment drugs.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical application technology. More specifically, it relates to the application of E3 ubiquitin ligase SIAH2 inhibitors in the preparation of renal protective drugs for lupus nephritis. Background Technology

[0002] Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by a loss of immune tolerance in the body's immune system. Patients produce large amounts of autoantibodies, leading to inflammatory responses and tissue dysfunction across multiple systems and organs. It is most prevalent in women of childbearing age (20-40 years old). The kidneys are one of the most common and primary organs affected by lupus, manifesting as lupus nephritis (LN), which is diagnosed via renal biopsy. LN patients are prone to progressing to end-stage renal disease (ESRD), leading to death and a series of social problems. Therefore, LN is a major cause of death and severe illness in SLE patients, and mitigating LN progression is a pressing challenge in SLE clinical diagnosis and treatment. Even with standardized immunotherapy, a large number of LN patients still develop irreversible renal fibrosis, and approximately 30% eventually develop ESRD, requiring dialysis or organ transplantation to sustain life. Renal fibrosis is a crucial step in the progression of LN to ESRD, marking the onset of irreversible kidney damage.

[0003] Currently, the clinical treatment of lupus nephritis (LN) typically involves medications such as hydroxychloroquine, rituximab, belimumab, corticosteroids, azathioprine, tacrolimus, cyclosporine, mycophenolate mofetil, and angiotensin-converting enzyme inhibitors. While these drugs are highly effective for LN, they also have certain side effects, including osteoporosis, obesity, elevated blood sugar, leukopenia, hair loss, loss of appetite, nausea, and vomiting. To reduce these side effects, there is still a lack of effective treatments for LN; therefore, there is an urgent need to develop more and safer new drugs for the treatment of LN. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the defects and deficiencies of the above-mentioned problems and to provide a new application of E3 ubiquitin ligase SIAH2 inhibitor in the preparation of nephropathy protective drugs for lupus nephritis.

[0005] The above-mentioned objective of this invention is achieved through the following technical solution:

[0006] This invention demonstrates that the SIAH2-LATS2 axis is involved in the pathogenesis of renal fibrosis in lupus nephritis (LN), revealing a novel mechanism by which SIAH2-LATS2 participates in LN renal fibrosis and confirming that LATS2 can serve as a target for preventing fibrosis progression in LN. This invention shows that LATS2 is abnormally downregulated in LN, and this downregulation is related to TGF-β-stimulated LATS2 ubiquitination and degradation mediated by the Zyxin-SIAH2-LATS2 signaling axis. SIAH2 causes LATS2 downregulation; therefore, using an E3 ubiquitin ligase SIAH2 inhibitor can restore LATS2 levels, reduce the production of the LATS2 downstream gene CTGF, and thus reduce further fibrotic damage in LN. This invention also shows that vitamin K3, as a SIAH2 inhibitor, can restore LATS2 levels, reduce CTGF production, reduce the occurrence of interstitial fibrosis, and reduce 24-hour proteinuria in LN, thus protecting kidney function and delaying LN progression.

[0007] Therefore, the present invention provides the use of E3 ubiquitin ligase SIAH2 inhibitors in the preparation of medicaments for the prevention and / or treatment of systemic lupus erythematosus, in the preparation of medicaments for alleviating the progression of lupus nephritis, in the preparation of medicaments for reducing kidney damage in lupus nephritis, or in the preparation of medicaments for alleviating kidney fibrosis in lupus nephritis.

[0008] Specifically, this invention reveals a novel mechanism by which SIAH2-LATS2 participates in renal fibrosis in lupus nephritis and confirms that LATS2 can serve as a target for preventing fibrosis progression in lupus nephritis. Therefore, any E3 ubiquitin ligase SIAH2 inhibitor that can inhibit SIAH2 and restore LATS2 levels can alleviate kidney damage, protect kidney function, and delay LN progression by restoring LATS2. As a preferred embodiment, this invention investigated the effect of vitamin K3 as an E3 ubiquitin ligase SIAH2 inhibitor on renal fibrosis in lupus nephritis. It also showed that vitamin K3 can alleviate lupus nephritis by acting on the SIAH2-LATS2 axis. Therefore, any E3 ubiquitin ligase SIAH2 inhibitor that can restore LATS2 levels can be used to prepare renal protective drugs for lupus nephritis.

[0009] Furthermore, the E3 ubiquitin ligase SIAH2 inhibitor restores LATS2 levels via the SIAH2-LATS2 axis.

[0010] Preferably, the E3 ubiquitin ligase SIAH2 inhibitor is vitamin K3.

[0011] Furthermore, the reduction refers to alleviating proteinuria levels and lowering the urine protein / creatinine ratio.

[0012] Furthermore, the relief is achieved by inhibiting the expression of CTGF and Collagen I, reducing fibrin production, and inhibiting collagen fiber deposition.

[0013] Furthermore, the relief refers to reducing the occurrence of interstitial fibrosis.

[0014] Furthermore, the dosage form of the drug is a capsule, tablet, oral preparation, microcapsule preparation, or injection.

[0015] The present invention has the following beneficial effects:

[0016] This invention provides a novel application of E3 ubiquitin ligase SIAH2 inhibitors in the preparation of renal protective drugs for lupus nephritis. This study shows that the SIAH2-LATS2 axis is involved in the pathogenesis of renal fibrosis in lupus nephritis; in lupus nephritis, LATS2 is significantly downregulated, leading to downstream YAP nuclear translocation and transcriptional activity, which mediates increased CTGF production and accelerates the progression of renal fibrosis. E3 ubiquitin ligase SIAH2 inhibitors can alleviate LATS2 downregulation and reduce CTGF production, thereby reducing renal fibrosis damage and protecting renal function. This invention repurposes an existing drug, using the E3 ubiquitin ligase SIAH2 inhibitor vitamin K3 to alleviate the transition of lupus nephritis to ESRD, providing a new option for mitigating the fibrotic progression of lupus nephritis. E3 ubiquitin ligase SIAH2 inhibitors can serve as effective drugs for treating lupus nephritis, with significant application value and significance in reducing renal fibrosis and preventing the progression of ESRD. Attached Figure Description

[0017] Figure 1 Figure 1 shows the results of immunohistochemical qualitative detection of LATS2 expression (A: kidney tissue sample from a patient with lupus nephritis; B: kidney tissue sample from a mouse with lupus nephritis; C: LATS2 expression in the renal cortical tubules).

[0018] Figure 2 The results are shown in the figures: Masson trichrome staining (A), Western spectroscopy to detect the effect of LATS2 knockdown on fibrosis factors (B), and real-time quantitative PCR to detect the expression of fibrosis-related genes (C). The results of the changes in the mRNA levels of various fibrosis-related factors (CTGF, α-SMA, COLAIV, TGF-β), CYR61 protein, ANKRD1 protein, Fibronectin, YAP gene and LATS2 at 12 weeks (D).

[0019] Figure 3 The image shows the results of immunohistochemical qualitative detection of SIAH2 and Zyxin expression.

[0020] Figure 4The results of Western blotting on the effect of TGF-β on LATS2 (A) and the interaction between Zyxin-SIAH2-LATS2 (BC) are shown in the figure.

[0021] Figure 5 The results are shown in Figure 1 (A) for the tissue localization of LATS2 and CTGF detected by fluorescence confocal microscopy and Figure 2 (B) for the expression of the target protein in the tissue by Western spectroscopy.

[0022] Figure 6 The results of Western blot analysis of the effect of VK3 on LATS2 levels (AB) and mouse urinary biochemical parameters (CD) are shown in the figure.

[0023] Figure 7 Image showing the results of HE and Masson staining in mouse kidney tissue pathology;

[0024] Figure 8 The image shows the results of immunohistochemical detection of LATS2 expression after Vitamin K3 treatment;

[0025] Figure 9 The images show the results of immunohistochemical qualitative detection of connective tissue growth factor (CTGF) expression and qualitative detection of type I collagen (Collagan I) expression after Vitamin K3 treatment.

[0026] Figure 10 The results of Western blot (A) and real-time quantitative PCR detection (B) of fibrosis-related genes after Vitamin K3 treatment are shown in the figure.

[0027] Figure 11 Image showing the results of Sirius red staining. Detailed Implementation

[0028] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0029] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0030] The following examples used β-cyclodextrin (purchased from Shanghai Aladdin), vitamin K3 (purchased from Shanghai Taoshu Biotechnology), and prednisolone (PNS, purchased from Shanghai Taoshu Biotechnology).

[0031] Example 1: Model Construction and Analysis

[0032] Two normal human kidney tissue samples and eight kidney tissue samples from patients with lupus nephritis were used in this embodiment, all collected from the First Affiliated Hospital of Jinan University.

[0033] Cell model: A tubular cell fibrosis model can be constructed by treating normal human renal tubular epithelial cells (HK-2) with TGF-β (20 ng / ml, purchased from Peprotech) for 48 hours.

[0034] Animal model: Eighteen MRL / lpr mice (purchased from Shanghai Slack), 4 weeks old, were used. Kidney tissue was collected from the mice at different disease stages (6, 8, and 12 weeks of feeding) for examination. A portion of the cortex from the fresh tissue was used for protein and RNA extraction, followed by routine Western blotting and real-time quantitative PCR analysis.

[0035] Kidney tissues from mice in each group were fixed with 4% paraformaldehyde to prepare 4μm continuous paraffin sections. The paraffin sections were cleared with xylene, hydrated with ethanol, and then stained with Masson's trichrome to observe pathological changes in the kidney tissue. In Masson's trichrome staining, extracellular blue represents fibrotic lesions. For immunohistochemical staining (IHC), paraffin sections (human and mouse samples) underwent microwave antigen retrieval and pore-breaking treatment with 0.1% Triton-100 after hydration. Anti-LATS2 antibody (purchased from Novus), SIAH2 antibody (purchased from Santa Cruz), and Zyxin antibody (purchased from Santa Cruz) were used to detect the expression of LATS2, SIAH2, and Zyxin, respectively. Immunohistochemical staining was performed according to the relevant kit procedures. A positive result was indicated by the appearance of brownish-yellow or brownish-brown granules in the cytoplasm under a light microscope. In laser confocal microscopy analysis, after primary antibody labeling, tissue sections are labeled with a matching fluorescently labeled secondary antibody (purchased from Abcam), then mounted with a DAPI-containing anti-fluorescence quenching mounting medium (purchased from Beyotime), and photographed.

[0036] The results are as follows Figure 1 As shown, in kidney tissue samples from patients with lupus nephritis ( Figure 1 A) and kidney tissue samples from mice with lupus nephritis ( Figure 1 In B), significant downregulation of LATS2 in the renal cortical tubules was observed (Figure 1C).

[0037] Masson staining results are as follows: Figure 2 As shown in Figure A, fibrotic lesions only appeared in MRL / lpr mice at 12 weeks of age. In vitro experiments showed that knocking down LATS2 levels led to decreased YAP phosphorylation and increased production of downstream connective tissue growth factor (CTGF). Figure 2 B, accelerates fibrosis. Figure 2C showed that TGF-β was upregulated at week 8. Figure 2 D shows that at week 12, various fibrosis-related factors (CTGF, α-SMA, COLA IV, TGF-β) were upregulated, and downstream genes of YAP (CTGF) were also upregulated, while LATS2 mRNA levels remained unchanged. Combining these results, it can be concluded that the downregulation of LATS2 (at week 8) precedes the onset of fibrosis (at week 12) and is synchronized with the increase in TGF-β levels, but is independent of LATS2 mRNA levels.

[0038] Since the mRNA level of LATS2 remained unchanged throughout the course of LN, it may be related to post-transcriptional modification. Further immunohistochemical staining revealed the following results: Figure 3 As shown, cells highly expressing SIAH2 and Zyxin were observed in the tubules of mice with lupus nephritis.

[0039] Furthermore, Western blot experiments showed that the downregulation of LATS2 was indeed caused by ubiquitination degradation induced by TGF-β stimulation. Figure 4 A). Zyxin upregulation and LATS2 downregulation further lead to increased YAP protein transcriptional activity (reduced phosphorylated YAP), resulting in a significant increase in downstream CTGF production, exacerbating fibrosis. Figure 4 B). Zyxin-SIAH2-LATS2 is a publicly disclosed ternary complex in which Zyxin acts as a scaffold protein to bring SIAH2 and LATS2 closer together, and SIAH2 acts as an E3 ubiquitin ligase to degrade LATS2, leading to downregulation of LATS2. Figure 4 C).

[0040] Laser confocal microscopy analysis revealed high CTGF expression in tubules with low LATS2 expression. Figure 5 A). Proteins were extracted from mouse kidney tissue, subjected to polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, incubated with primary antibody, labeled with secondary antibody, and imaged using an automated imaging system to detect the expression levels of LATS2, YAP, p-YAP, GAPDH, CTGF, and SIAH2 proteins. Results are as follows: Figure 5 B showed upregulation of SIAH2 and Zyxin, downregulation of LATS2, decreased YAP phosphorylation, and increased downstream CTGF in mouse kidney tissue.

[0041] Example 2: Pharmacological Experiment

[0042] A TGF-β-induced HK-2 cell fibrosis model was used, and the cells were treated with vitamin K3, an inhibitor of the E3 ubiquitin ligase SIAH2. The expression of LATS2 was detected by Western blotting.

[0043] Eighteen 8-week-old MRL / lpr mice were randomly divided into three groups (Vehicle negative control group, Vitamin K3 group, and PNS positive control group) after 7 days of acclimatization. Six mice were assigned to each group. C57 / BL6 mice were also included as the WT group. The Vehicle control group received β-cyclodextrin, the Vitamin K3 group received Vitamin K3 (10 mg / kg) formulated with β-cyclodextrin, and the PNS control group received prednisolone (PNS) (2 mg / kg) formulated with β-cyclodextrin. Mice in all four groups were sacrificed after 4 weeks of treatment, and kidney tissue was collected for examination, following the same methods as in Example 1.

[0044] Biochemical parameters and urine / creatinine ratio were measured in mouse urine samples. Mice were allowed free access to water in metabolic cages and were fasted. Urine samples were collected from mice over 24 hours, and urine biochemical parameters were measured using a urine protein and creatinine detection kit (Nanjing Jiancheng Biotechnology).

[0045] The results are as follows Figure 6 As shown, Vitamin K3 treatment reduced the degradation of LATS2 in cells ( Figure 6 A). Simultaneously, Vitamin K3 treatment also led to enhanced YAP phosphorylation and reduced CTGF (…). Figure 6 B). In animal models, biochemical results from mouse urine samples showed that Vitamin K3 significantly alleviated proteinuria levels (B). Figure 6 C) and reduced the urine protein / creatinine ratio in mice ( Figure 6 D). This indicates that Vitamin K3 can reduce kidney damage in lupus nephritis.

[0046] Example 3: Morphological observation of kidney tissue

[0047] Kidney tissues from mice in each group treated with the drug in Example 2 were fixed with 4% paraformaldehyde. The tissues were then dehydrated using a routine gradient of ethanol, cleared with xylene, embedded in paraffin, and sectioned into 4μm serial paraffin sections. Histopathological changes in the kidney tissues were observed using HE and Masson staining.

[0048] Mouse kidney histopathology results with HE and Masson staining are as follows: Figure 7 As shown, the lupus nephritis model exhibited significant fibrosis (abundant blue collagen deposition). After treatment with the SIAH2 inhibitor Vitamin K3, VK3 reduced interstitial fibrosis, and renal fibrosis was significantly improved (reduced blue collagen deposition).

[0049] The expression of LATS2 was then qualitatively detected by immunohistochemistry, following the same procedure as in Example 1. The results are as follows: Figure 8As shown, treatment with the SIAH2 inhibitor Vitamin K3 significantly upregulated LATS2 levels. Immunohistochemical qualitative analysis of the expression of pro-fibrotic factors CTGF and Collagen I yielded the following results: Figure 9 As shown, Vitamin K3 treatment significantly reduced the expression of CTGF and Collagen I, and reduced fibrin production.

[0050] Further Western blot and real-time quantitative PCR detection were performed on the tissues, using the same methods as in Example 1. The results are as follows: Figure 10 As shown, treatment with Vitamin K3 significantly upregulated LATS2 levels and inhibited the expression level of the fibrosis marker CTGF. Figure 10 A), upregulation of LATS2 reduces CTGF mRNA levels and decreases other fibrosis genes such as Collagen I. Figure 10 The expression level of B) can alleviate fibrosis.

[0051] Finally, the results were obtained by staining with Sirius red. Figure 11 As shown, treatment with Vitamin K3 significantly inhibits collagen fiber deposition (red) and reduces interstitial fibrosis.

[0052] In summary, this invention, in its study of the mechanism of kidney injury in lupus nephritis, found that SIAH2-mediated LATS2 degradation is a crucial step in the progression of lupus nephritis to renal fibrosis. LATS2 is abnormally downregulated in the lupus nephritis (LN), and this downregulation is associated with TGF-β-stimulated Zyxin-SIAH2-LATS2-mediated LATS2 ubiquitination and degradation. Restoring LATS2 levels can reduce the production of the downstream gene CTGF, thereby reducing further fibrotic damage in lupus nephritis. Therefore, the SIAH2-LATS2 axis can serve as a target for preventing fibrosis progression in lupus nephritis. Since SIAH2 induces LATS2 downregulation, using an E3 ubiquitin ligase SIAH2 inhibitor can restore LATS2 levels and alleviate kidney injury through LATS2. This study shows that vitamin K3, as an inhibitor of the E3 ubiquitin ligase SIAH2, can reduce the degradation of LATS2 mediated by TGF-β, restore LATS2 levels, reduce the production of CTGF, reduce the occurrence of interstitial fibrosis, significantly alleviate fibrotic damage in LN mice, reduce 24-hour proteinuria in lupus nephritis, and play a role in protecting kidney function and delaying the progression of LN.

[0053] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of E3 ubiquitin ligase SIAH2 inhibitors in the preparation of drugs to alleviate the progression of lupus nephritis, characterized in that, The E3 ubiquitin ligase SIAH2 inhibitor is vitamin K3.

2. The application of E3 ubiquitin ligase SIAH2 inhibitors in the preparation of drugs to alleviate kidney damage in lupus nephritis, characterized in that, The E3 ubiquitin ligase SIAH2 inhibitor is vitamin K3.

3. The application of E3 ubiquitin ligase SIAH2 inhibitors in the preparation of drugs to alleviate renal fibrosis in lupus nephritis, characterized in that, The E3 ubiquitin ligase SIAH2 inhibitor is vitamin K3.

4. The application according to any one of claims 1 to 3, characterized in that, The E3 ubiquitin ligase SIAH2 inhibitor restores LATS2 levels via the SIAH2-LATS2 axis.

5. The application according to claim 2, characterized in that, The relief refers to alleviating proteinuria levels and reducing the urine protein / creatinine ratio.

6. The application according to claim 3, characterized in that, The relief is achieved by inhibiting the expression of CTGF and Collagen I, reducing fibrin production, and inhibiting collagen fiber deposition.

7. The application according to claim 3, characterized in that, The relief refers to reducing the occurrence of interstitial fibrosis.

8. The application according to any one of claims 1 to 3, characterized in that, The dosage form of the drug is capsules, tablets, or injections.

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