Application of NETs inhibitor DNase I in treatment of IgA nephropathy

Abstract

The invention discloses application of an NETs inhibitor DNase I in treatment of IgA nephropathy, and belongs to the technical field of biological medicines. NETs is taken as a treatment target, and a DNA skeleton in the NETs is specifically degraded through DNase I, so that immune complex deposition in an IgAN glomerular mesangial region is reduced, expression of proinflammatory factors IL-1beta, TNF-alpha and IL-6 is inhibited, and glomerular pathological injury and renal function indexes are improved. According to the invention, a precise treatment strategy based on NETs inhibition is provided by targeted removal of NETs-mediated pathological cascade reaction.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an application of a NETs inhibitor DNase I in the treatment of IgA nephropathy. Background Art

[0002] IgA nephropathy (IgAN) is a chronic immune glomerular disease characterized by the deposition of IgA1 immune complexes in the glomerular mesangium. Clinical manifestations include hematuria, proteinuria, and progressive renal impairment, with some patients progressing to end-stage renal disease. Its pathogenesis remains incompletely understood, but it involves a multifactorial interaction involving genetic susceptibility, mucosal immune abnormalities, and complement system activation. Traditional treatment focuses on renin-angiotensin system inhibitors (ACEIs / ARBs), supplemented with glucocorticoids to control the disease in high-risk patients. However, approximately 30%-40% of patients experience suboptimal responses and ultimately require renal replacement therapy. Therefore, further investigation of the pathogenic factors that contribute to the development and progression of IgA nephropathy is needed to provide new insights into clinical intervention and treatment.

[0003] Neutrophil extracellular traps (NETs) are secreted by activated neutrophils and consist of a network of double-stranded DNA (dsDNA), myeloperoxidase (MPO), citrullinated histone 3 (citH3), and antimicrobial proteins. They are responsible for capturing and killing extracellular pathogens. Their degradation primarily relies on the cooperative action of extracellular DNases (DNases). Excessive release of NETs can promote immune inflammatory responses. DNase I is a DNA-specific endonuclease that hydrolyzes DNA. As a NET inhibitor, DNase I acts by degrading the DNA backbone within NETs, thereby disrupting their structure. This may reduce NET-mediated inflammation and tissue damage. NETs are implicated in the pathogenesis of various autoimmune and inflammatory diseases, such as systemic lupus erythematosus, rheumatoid arthritis, atherosclerosis, and malignant tumors. However, the relationship between NETs and IgA nephropathy remains unclear, and the NET inhibitor DNase I has not been used in IgA nephropathy. Summary of the Invention

[0004] The present invention provides use of a NETs inhibitor DNase I in the treatment of IgA nephropathy.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0006] Application of NETs inhibitor DNase I in IgA nephropathy.

[0007] Furthermore, DNase I specifically degrades the DNA skeleton of NETs, thereby destroying its structural integrity and blocking the pathological cascade reaction mediated by NETs.

[0008] Application of DNase I in preparing a pharmaceutical composition for treating IgA nephropathy.

[0009] Furthermore, the DNase I reduces the deposition of NETs markers CitH3 and MPO in the glomerular mesangial area.

[0010] Furthermore, the DNase I reduces the deposition of IgA / IgG / C3 immune complexes in the glomerular mesangial area.

[0011] Furthermore, the DNase I downregulates the mRNA expression of pro-inflammatory factors IL-1β, TNF-α, and IL-6, thereby alleviating renal inflammatory response.

[0012] Furthermore, the DNase I improves glomerular pathological damage and renal function indicators.

[0013] Application of NETs inhibitor DNase I in improving the deposition of immune complexes in the glomerular mesangial area.

[0014] Compared with the prior art, the present invention has the following advantages:

[0015] This invention reveals for the first time the therapeutic potential of DNase I in IgAN by specifically inhibiting the pathological effects of NETs, opening up new directions for disease mechanism research and clinical intervention. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0017] Figure 1 Figure 3 is the relationship between NETs and the onset of IgAN; (A) dsDNA levels in the serum of IgAN patients and healthy subjects; (B) CitH3 levels in the serum of IgAN patients and healthy subjects; (C) Immunofluorescence images of kidney tissues of IgAN and WT mice, scale = 20 μm; (D) Representative western blot images of CitH3 and MPO protein expression in kidney tissues of IgAN and WT mice; (E) Quantitative analysis of CitH3 and MPO protein expression; *: P < 0.05; **: P < 0.01; ***: P < 0.001.

[0018] Figure 2 Schematic diagram of the results showing that the NETs inhibitor DNase I can significantly improve the renal function of IgAN mice; (A) serum Scr, (B) serum BUN, (C) urine albumin, and (D) serum sIgA test results of mice in each group; *: P<0.05; **: P<0.01; ***: P<0.001.

[0019] Figure 3 These are the HE and PAS staining images of kidney tissues of mice in each group, scale bar = 25 μm.

[0020] Figure 4 (A) IgA (B) IgG (C) C3 immunofluorescence images and (D-F) quantitative analysis images of kidney tissues of mice in each group, scale bar = 25 μm, *: P < 0.05; **: P < 0.01; ***: P < 0.001.

[0021] Figure 5 The relative expression levels of inflammatory indicators (A) IL-1β, (B) TNF-α, and (C) IL-6 mRNA in the kidney tissues of mice in each group, *: P<0.05; **: P<0.01; ***: P<0.001. DETAILED DESCRIPTION

[0022] To gain a deeper understanding of the present invention, we will provide a comprehensive and detailed description thereof. However, the present invention has various implementations and is not limited to the specific examples listed herein. These examples are presented to enhance a comprehensive understanding of the present disclosure.

[0023] Example 1

[0024] (1) Experimental methods and steps:

[0025] ① Serum dsDNA levels in IgAN patients and healthy subjects were measured using the Quant-iT™ PicoGreen™ dsDNA Assay Kit. Human citrullinated histone H3 (CitH3) ELISA assay kit was used to quantify CitH3 levels in serum from IgAN patients and healthy subjects according to the manufacturer's instructions.

[0026] ② Experimental Animals and Grouping: Sixteen male IgAN mice were provided by the Central Laboratory of Shanxi Provincial People's Hospital. All IgAN model mice were bred from miRNA-23b-3p knockout mice (donated by the Center for Precision Medicine in Kidney Diseases, Beihua University). They were housed in an SPF-grade animal facility. The IgAN mice were randomly divided into two groups: an IgAN group (n=8) and an IgAN+DNase I group (n=8). Wild-type littermates served as a control group (WT) (n=8). Mice in the IgAN+DNase I group received intraperitoneal injections of the NETs inhibitor DNase I (5 mg / kg / day). Mice in the IgAN+DNase I group received normal saline for four consecutive weeks.

[0027] ③ Detection of mouse renal function-related indicators: Collect the urine of mice 24 hours before sacrifice in a metabolic cage, and measure the amount of mouse urine albumin in 24 hours according to the instructions of the commercial kit; obtain mouse serum samples and measure the levels of mouse serum BUN and Scr according to the instructions of the kit.

[0028] ④ According to the manufacturer's instructions, the mouse secretory immunoglobulin A (sIgA) ELISA assay kit was used to quantify the sIgA level in the serum of each group of mice.

[0029] ⑤ Histological staining: After mice were sacrificed, renal tissue was isolated, fixed with 4% paraformaldehyde, embedded in paraffin, and cut into 4-μm-thick sections. Sections were stained with hematoxylin and eosin (HE) and periodic acid-Schiff (PAS). Twenty fields of view were randomly selected for observation of renal pathological changes and imaging of renal tissue.

[0030] ⑥ Protein extraction and Western blotting: 15 mg of mouse kidney was collected and 200 μL of RIPA lysis buffer was added to extract total kidney protein. Equal amounts of protein were separated by SDS-PAGE, wet-transferred, and blocked with 5% skim milk for 1.5 h. The membranes were then incubated with CitH3 (1:1000), MPO (1:3000), and β-actin (1:10,000) antibodies, respectively. The membranes were incubated overnight at 4°C, washed with TBST (three times, 10 min each), incubated with secondary antibodies at room temperature for 1 h, and washed with TBST (three times, 5 min each). The membranes were developed using electrochemiluminescence (ECL) solution. Finally, images were taken using an automated gel imaging system, and grayscale analysis was performed using Image J software.

[0031] ⑦ Immunofluorescence staining: Frozen sections of kidney tissue from IgAN mice and normal control mice were co-stained with CitH3-FITC and MPO-Cy3 to observe the levels of NETs in the kidney tissue. In addition, frozen sections of kidney tissue from mice in each group were immunostained with IgA, IgG, and complement C3 to observe the level of immune complex deposition in mice.

[0032] ⑧RT-qPCR determination of mouse kidney mRNA expression: Mouse kidney tissue was quickly frozen in liquid nitrogen and then ground. Total RNA was extracted with Trizol and the purity was tested. 1-2 μg of RNA was used as a template for cDNA synthesis using reverse transcriptase. Specific primers for IL-1β, TNF-α, and IL-6 were designed and synthesized. SYBR Green Mix was used to establish a qPCR reaction system. Internal reference genes and technical replicates were set. Amplification was performed after pre-denaturation at 95°C (denaturation-annealing / extension). Primer specificity was verified using melting curves, and the relative expression of target genes was calculated using the ΔΔCT method based on the Ct value (2 -ΔΔCT ).

[0033] 9. Statistical Methods: All data are expressed as mean ± standard deviation and analyzed using SPSS 22.0 software. One-way ANOVA was used for comparisons between multiple groups, and the LSD t-test was used for comparisons between two groups. Differences were considered statistically significant when P < 0.05. GraphPad Prism 8.0 software was used for plotting.

[0034] (2) Experimental results:

[0035] ①Relationship between NETs and the pathogenesis of IgAN

[0036] Analysis by Quant-iT™ PicoGreen™ dsDNA Assay Kit showed that ( Figure 1 A) The serum dsDNA level in IgAN patients was significantly higher than that in healthy controls (P<0.05). Figure 1 B) The serum CitH3 concentration in IgAN patients was significantly higher than that in the healthy control group (P<0.05). In addition, the kidney tissues of IgAN model mice and WT mice were immunofluorescently co-stained with NETs markers MPO and CitH3. Figure 1 As shown in C, compared with the WT group, the renal tissue of the IgAN group mice had obvious NETs deposition. Then, the expression levels of NETs marker proteins CitH3 and MPO in the renal tissue of mice were detected by Western blot. Figure 1 As shown in Figures D and E, compared with the WT group, the expression of NET marker proteins CitH3 and MPO in the kidney tissue of mice in the IgAN group was significantly increased (P < 0.05). These experimental results indicate that increased NET formation is closely related to the pathogenesis of IgAN.

[0037] ②NETs inhibitor DNase I can significantly improve renal function in IgAN mice

[0038] like Figure 2 As shown in the results, compared with the WT group, the serum levels of Scr, BUN, and urinary albumin in the IgAN group were significantly increased (P < 0.05). Treatment with the NET inhibitor DNase I significantly decreased Scr, BUN, and urinary albumin (P < 0.05). Furthermore, compared with the WT group, the serum levels of sIgA in the IgAN group were significantly increased. Treatment with the NET inhibitor DNase I significantly decreased sIgA levels (P < 0.05). These results indicate that the NET inhibitor DNase I can significantly improve renal function in IgAN mice.

[0039] ③ Treatment with NETs inhibitor DNase I can alleviate renal histopathological changes in IgAN mice

[0040] like Figure 3 As shown, compared with the WT group, HE staining in the IgAN model mice revealed glomerular structural disorder, mesangial cell proliferation, and inflammatory cell infiltration. PAS staining indicated basement membrane thickening and extensive deposition of PAS-positive substances (such as glycoproteins or immune complexes) in the mesangial region, consistent with the typical pathological features of IgA nephropathy. After DNase I treatment, HE staining showed a reduction in glomerular damage and inflammatory infiltration, and PAS staining significantly decreased PAS-positive deposits in the mesangial region. This suggests that DNase I may reduce immune complex deposition by degrading extracellular DNA, thereby ameliorating pathological damage in the renal tissue of IgAN mice. This result suggests that DNase I has a potential therapeutic effect on IgA nephropathy.

[0041] ④ Treatment with the NETs inhibitor DNase I can alleviate the changes in immune complex deposition in the glomerular mesangial region of IgAN mice

[0042] Depend on Figure 4 As shown in the figure, immunofluorescence results showed that compared with the WT group, the deposition of IgA, IgG, and C3 in the glomeruli of mice in the IgAN model group was significantly increased. After intervention with the NETs inhibitor DNase I, the deposition of IgA, IgG, and C3 was significantly reduced, and the differences were statistically significant (P<0.05). These results indicate that the NETs inhibitor DNase I has a significant effect on improving the deposition of immune complexes in the glomerular mesangium.

[0043] NETs inhibitor DNase I can reduce renal inflammation in IgAN mice

[0044] RT-qPCR was used to detect the relative expression levels of inflammatory cytokines IL-1β, TNF-α, and IL-6 mRNA in mouse kidney tissue. Figure 5As shown in the results, compared with the WT group, the mRNA expression levels of IL-1β, TNF-α, and IL-6 in the IgAN model mice were significantly increased (P < 0.05). After treatment with the NETs inhibitor DNase I, the mRNA expression levels of IL-1β, TNF-α, and IL-6 were significantly decreased (P < 0.05). These experimental results indicate that the NETs inhibitor DNase I can alleviate renal inflammation in IgAN mice.

[0045] Any matters not described in detail in this specification are prior art known to those skilled in the art. Although the above description of the present invention is based on specific embodiments to facilitate understanding of the present invention by those skilled in the art, it should be understood that the present invention is not limited to the scope of the specific embodiments. As long as various modifications are within the spirit and scope of the present invention as defined and determined by the appended claims, such modifications will be obvious to those skilled in the art, and all inventions and creations utilizing the concepts of the present invention are protected.

Claims

1. Application of NETs inhibitor DNase I in IgA nephropathy.

2. The use of the NETs inhibitor DNase I in IgA nephropathy according to claim 1, characterized in that: DNase I specifically degrades the DNA skeleton of NETs, thereby destroying its structural integrity and blocking the pathological cascade reaction mediated by NETs.

3. Application of DNase I in the preparation of a pharmaceutical composition for treating IgA nephropathy.

4. The use according to claim 3, characterized in that: The DNase I reduces the deposition of NETs markers CitH3 and MPO in the glomerular mesangial area.

5. The use according to claim 3, characterized in that: The DNase I reduces the deposition of IgA / IgG / C3 immune complexes in the glomerular mesangial area.

6. The use according to claim 3, characterized in that: The DNase I downregulates the mRNA expression of pro-inflammatory factors IL-1β, TNF-α, and IL-6, thereby alleviating kidney inflammatory response.

7. The use according to claim 3, characterized in that: The DNase I improves glomerular pathological damage and renal function indicators.

8. Application of NETs inhibitor DNase I in improving the deposition of immune complexes in the glomerular mesangial region.

Images