Application of reagent for detecting SRC and CYBB expression in preparation of product for diagnosing and treating lupus nephritis

Abstract

The invention relates to an application of a reagent for detecting SRC expression in preparation of a product for diagnosing and treating lupus nephritis, SRC is highly expressed in a patient with lupus nephritis, when the expression level of SRC is reduced, ferroptosis of podocyte is remarkably relieved, ferroptosis related markers are remarkably reversed, the levels of Fe < 2 + > and MDA are reduced, the level of GSH is increased, and compared with a control group, the SRC expression level is remarkably reduced. The viability of podocyte is increased; the invention relates to application of a reagent for detecting CYBB expression in preparation of a product for diagnosing and treating lupus nephritis, CYBB is highly expressed in a patient suffering from lupus nephritis, when the expression level of CYBB is reduced, ferroptosis in podocytes is remarkably relieved, ferroptosis related markers are remarkably reversed, the levels of ACSL4, Fe < 2 + >, ROS and MDA are reduced, and the levels of GPX4 and GSH are increased, so that the effect of treating lupus nephritis is achieved. The polarization of M2 macrophages is promoted, the ferroptosis phenomenon is further relieved through the polarization of the macrophages, and the viability of podocytes is improved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of reagents for detecting SRC and CYBB expression in the preparation of products for the diagnosis and treatment of lupus nephritis. Background Technology

[0002] Systemic lupus erythematosus (SLE) is a complex chronic autoimmune disease that can affect multiple systems and organs throughout the body. Studies show that 30% to 60% of adult SLE patients may develop lupus nephritis (LN), with the rate being even higher in children, reaching 70%. Lupus nephritis is not only an important clinical manifestation of SLE but also a major risk factor for chronic and end-stage renal disease, primarily due to glomerular inflammation and damage.

[0003] Ferroprelation is a unique form of cell death involving decreased glutathione peroxidase 4 (GPX4) activity and reduced ability to scavenge hydroperoxides of polyunsaturated fatty acids. Disorders of iron metabolism are thought to be associated with the activation of ferroprelation in the pathogenesis of lacrimal nephritis (LN). Although ferroprelation has been extensively studied in various diseases, its specific pathological mechanisms in LN remain unknown and require further investigation.

[0004] Macrophages are key regulators of the immune system, and their polarization between anti-inflammatory and pro-inflammatory states profoundly influences the progression of various diseases. Recent studies suggest that anti-inflammatory polarized macrophages in the lymphocyte nucleus (LN) may help reduce podocyte injury. Podocytes are an important component of the glomerular filtration barrier. However, the specific mechanisms by which macrophage polarization affects ferroptosis activation and podocyte injury in the LN remain unclear and require further investigation.

[0005] The mechanisms of podocyte injury are complex and diverse, including genetic factors, immune-mediated damage, and metabolic disorders. This damage can lead to the loss of podocyte foot processes, detachment from the glomerular basement membrane (GBM), and subsequent proteinuria, even glomerulosclerosis and other nephropathy. The protein encoded by the CYBB gene is a core component of the NADPH oxidase complex, playing a crucial role in oxidative stress across various cell types. In immune cells such as neutrophils and monocytes, NADPH oxidase is responsible for producing large amounts of reactive oxygen species to combat invading pathogens, constituting an important part of the innate immune system.

[0006] The main culprits of lupus nephritis (LN) are B cells, but current depletion therapies targeting B cells are often ineffective. Therefore, finding new treatment strategies for lupus nephritis has become a research focus. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention proposes the application of reagents for detecting SRC expression in the preparation of products for the diagnosis and treatment of lupus nephritis, and the application of reagents for detecting CYBB expression in the preparation of products for the diagnosis and treatment of lupus nephritis.

[0008] The technical solution of this invention is as follows: Application of reagents for detecting SRC expression in the preparation of products for the diagnosis and treatment of lupus nephritis.

[0009] Furthermore, the SRC is highly expressed in patients with lupus nephritis.

[0010] Furthermore, the product includes SRC detection reagents.

[0011] Furthermore, the detection reagents include those used to detect SRC expression levels via quantitative real-time PCR.

[0012] Application of reagents for detecting CYBB expression in the preparation of products for the diagnosis and treatment of lupus nephritis.

[0013] Furthermore, the CYBB is highly expressed in patients with lupus nephritis.

[0014] Furthermore, the product includes a CYBB detection reagent.

[0015] Furthermore, the detection reagents include those used to detect CYBB expression levels by quantitative real-time PCR.

[0016] Application of reagents for detecting SRC and CYBB expression in the preparation of products for the diagnosis and treatment of lupus nephritis.

[0017] Compared with the prior art, the present invention has at least the following advantages: 1. This invention relates to the application of reagents for detecting SRC expression in the preparation of products for the diagnosis and treatment of lupus nephritis, wherein SRC is highly expressed in patients with lupus nephritis, and when the expression level of SRC decreases, ferroptosis in podocytes is significantly alleviated, and ferroptosis-related markers are significantly reversed, and Fe... 2+ Decreased levels of ferroptosis-related (ferroptosis-related) and MDA (malondialdehyde, a marker of lipid peroxidation), increased levels of GSH (a negative regulator of ferroptosis), and increased podocyte viability compared to the control group.

[0018] 2. This invention relates to the application of reagents for detecting CYBB expression in the preparation of products for the diagnosis and treatment of lupus nephritis. CYBB is highly expressed in patients with lupus nephritis. When the expression level of CYBB decreases, ferroptosis in podocytes is significantly alleviated, and ferroptosis-related markers are significantly reversed, including ACSL4 (ferroptosis-associated protein) and Fe... 2+The levels of ROS and MDA (a marker of lipid peroxidation) decreased, while the levels of GPX4 (a negative regulator of ferroptosis) and GSH (a negative regulator of ferroptosis) increased. This also promoted the polarization of M2 macrophages, which further alleviated ferroptosis and increased the activity of podocytes. Attached Figure Description

[0019] To more clearly illustrate the specific embodiments of the present invention, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below.

[0020] Figure 1 Example 1: Bioinformatics mining of differentially expressed genes in patients with lupus nephritis; Figure 2 Example 2: Analysis of the relationship between CYBB high expression and podocyte ferroptosis; Figure 3 For the pathological characteristics analysis of the kidney LN in MRL / lpr mice in Example 3; Figure 4 Example 4: CYBB participates in regulating ferroptosis in LN podocytes; Figure 5 Example 5 shows a positive correlation between CYBB expression and macrophage M1 polarization; Figure 6 Example 6: CYBB-mediated ferroptosis induces increased M1 polarization in macrophages; Figure 7 Example 7: CYBB and SRC synergistically promote ferroptosis in LN podocytes. Detailed Implementation

[0021] The present invention will now be described in further detail. It should be noted that the following specific embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above application content.

[0022] This invention provides a general and / or specific description of the materials and experimental methods used in the experiments. Unless otherwise specified, all experimental or testing methods are conventional methods; all reagents or instruments used, unless otherwise specified, are commercially available conventional products prepared or used using conventional methods.

[0023] Source of materials: MRL / MpJ mice: Skebers.

[0024] Method for constructing MRL-lpr (LN) mice: 6-8 week old BALB / c (or C57BL / 6) female mice (females are more likely to induce lupus-like lesions than males) were selected and injected intraperitoneally with 0.5 mL of norphyrane at a concentration of 0.783 g / ml.

[0025] Experimental methods Collection of GSE datasets and ferroptosis-related genes (FRGs): Gene expression data and related clinical information were obtained from the GEO database (https: / / www.ncbi.nlm.nih.gov). The focus was on "lupus nephritis," with the search limited to "glomeruli or tubulointerstitial." In the selection process, particular attention was paid to humans (Homo sapiens). The GSE72326 microarray dataset was selected based on the above criteria. This dataset includes kidney biopsy samples from lupus nephritis (LN) and kidney samples from healthy living donors. These samples were further subdivided into glomerular and tubulointerstitial portions for in-depth microarray analysis. In addition, this embodiment also used the GSE113342 microarray dataset based on the nCounter Nanostring Human Immunology v2 platform, covering 504 immune-related genes. This dataset provides clinical information, including age, pathological classification, and 12-month treatment response, which is crucial for validating ferroptosis-related genes (FRGs) and their relationship with clinical characteristics. The set of ferroptosis-associated genes (FRGs) consisted of 275 protein-coding genes downloaded from FerrDb and the GeneCards database (https: / / www.genecards.org / ).

[0026] Identification of differentially expressed genes (DEGs): Microarray data analysis was performed using the "limma" package in R. DEGs from LN and healthy donors were identified in RStudio (version 13.1093). DEGs with adjusted p-values ​​<0.05 and log2FC >0.58 were defined as statistically significant upregulated or downregulated genes. Volcano plots of DEGs were created using the R package "ggplot2". Subsequently, the intersection of DEGs and FRGs (ferroptosis-associated genes) was analyzed using the R package "VennDiagram", defining them as ferroptosis-associated DEGs (FR-DEGs) and representing them in a Venn diagram.

[0027] Cell viability assay: Primary podocytes isolated from MRL / MpJ (control) and MRL-lpr (LN) mice were subjected to cell viability assay at 5 × 10⁻⁶ cells / mL. 3Cells were seeded at a density of 10 cells / well in 96-well plates to ensure good adhesion and growth. CCK-8 reagent was mixed with culture medium at a specific ratio, typically 10 μl CCK-8 solution to 100 μl of culture medium. The mixed CCK-8 solution was added to each well, typically 10 μl per well. The 96-well plates were incubated for 1 hour. After incubation, the absorbance (OD value) of each well was measured using a microplate reader at 450 nm. Cell viability was calculated based on the absorbance values, with the absorbance of the control group set as 100%. The absorbance values ​​of the experimental groups were compared with those of the control group to calculate changes in cell viability. The experimental results were analyzed to determine the effect of the test substance on cell proliferation or cytotoxicity.

[0028] CFDA staining: Primary cultured podocytes were stained with pre-warmed 5μM CFDA-PBS for 15 minutes. After the culture medium was replaced with fresh medium, the cells were observed under a fluorescence microscope (excitation wavelength 485nm) within 30 minutes. Cells that showed bright green fluorescence were considered to be alive.

[0029] Transmission electron microscopy (TEM): Kidney tissue for electron microscopy observation was fixed with 3% glutaraldehyde and 1% osmium tetroxide (OsO4), then dehydrated by a series of gradient ethanol and washed with acetone. The samples were then embedded in Epon 812 embedding resin, ultrathin sections were stained with uranium acetate and lead citrate, and examined using a transmission electron microscope (JEM-1230; JEOL, Japan).

[0030] Western blotting: Total protein was extracted from cell samples, and protein concentration was determined using the BCA (biuret) method. For Western blotting analysis, three replicate samples were selected from each group. Western blotting was performed in two stages: initially run at a constant voltage of 80V for 20 minutes, then increased to 120V until electrophoresis was complete. Proteins were then transferred to the membrane at a constant current of 200mA for 90 minutes. The membrane was blocked with 5% skim milk powder at room temperature for 1 hour. After blocking, the membrane was incubated with primary antibody overnight at 4°C. Primary antibodies included: iNOS antibody (ab3523, Abcam), CD206 antibody (81525-1-RR, Proteintech), NOX2 (CYBB) antibody (PA5-79118, Invitrogen), SRC antibody (AHO1152, Invitrogen), ACSL4 / FACL4 antibody (81196-1-RR, Proteintech), GXP4 antibody (ab125066, Abcam), and GAPDH antibody (MA5-15738, Invitrogen). After incubation with primary antibodies, the membrane was washed and incubated with secondary antibodies on a shaker at room temperature for 1 hour. After incubation with secondary antibodies, the membrane was washed again. Protein bands were visualized using ECL (enhanced chemiluminescence) assay reagents, and chemiluminescence signals were captured on the film using a chemiluminescence imaging system. The optical density of the protein bands was quantified using ImageJ software.

[0031] Statistical Analysis: Statistical analysis of the experiments was performed using GraphPad Prism version 9 (GraphPad Software). Significance was determined by one-way ANOVA and Tukey post-hoc analysis, with a p-value < 0.05. All data are expressed as mean ± standard deviation (SD).

[0032] Gene knockdown and overexpression: Transient knockdown of CYBB and SRC was performed by transfection with sh-CYBB and sh-SRC plasmids (ribobio, China). Transient overexpression of CYBB was performed by transfection with the CYBB plasmid (ribobio, China). Transfection was performed using Lipofectamine 2000. Cells were collected 24 hours after transfection for further analysis to evaluate the efficiency of knockdown / overexpression.

[0033] Co-immunoprecipitation (Co-IP): LN podocytes were lysed with TNE buffer (10 mM Tris-HCl, pH 7.4, 1% Nonidet P-40, 1 mM EDTA) and pre-incubated with protein A / G magnetic beads for 4 hours. The pre-incubated beads were removed by magnetic separation to eliminate non-specific binding, and the pre-cleaned lysate was collected. The lysate was then immunoprecipitated overnight with magnetic beads conjugated with either anti-CYBB antibody (PA5-79118, Invitrogen) or anti-SRC antibody (MA5-47738, Invitrogen). The beads were washed four times with TNE buffer and eluted with SDS sample buffer. The eluted proteins were detected by Western blot using the corresponding antibodies.

[0034] HE staining: Tissues are fixed in 10% neutral buffered formaldehyde and then dehydrated using a gradient of ethanol solutions. After dehydration, the tissues are cleared with xylene or a similar solvent to remove residual alcohol. The tissues are then embedded in a medium such as paraffin or plastic for sectioning. Typically, 4-6 micrometer thick slices are cut from the embedded blocks, mounted on slides, and allowed to dry. Dewaxing is then performed by treating the slides with xylene or a substitute to remove paraffin. Rehydration is completed by treating the slides in a series of decreasing concentrations of alcohol to water. The staining process begins with hematoxylin staining to make the nuclei blue, followed by differentiation using solutions such as water or acidic alcohol to remove excess dye. Eosin staining is then used to make the cytoplasm red. After staining, the slides are dehydrated and cleared again, and finally mounted using a medium such as Canada balsam for preservation. Finally, the stained tissue sections are observed under an optical microscope to assess cell and tissue morphology.

[0035] RT-qPCR: Total RNA was extracted from podocytes and its concentration was measured. RNA was reverse transcribed into cDNA using the PrimeScript RT kit (TaKaRa). Quantitative real-time PCR was performed using the SYBR Green kit (TaKaRa), and mRNA expression levels were normalized to GAPDH using the 2-ΔΔCt method. The forward and reverse primer sequences are as follows: CCL2: 5′-AGCAAGATGATCCCAATGAGT-3′ (SEQ ID NO.1) and 5′-GAGCTTGGTGACAAAAACTACAG-3′ (SEQ ID NO.2) CCR2: 5′-CAAAATGGATGCCTTAGCACTG-3′ (SEQ ID NO.3) and 5′-CCAGGTTTTCATGTTTGGCTATTC-3′ (SEQ ID NO.4) GAPDH: 5′-CAGAACATCATCCCTGCATC-3′ (SEQ ID NO.5) and 5′-CTGCTTCACCACCTTCTTGA-3′ (SEQ ID NO.6) Immunofluorescence analysis: Coverslips were washed with PBS and fixed with 4% paraformaldehyde for 30 minutes. Each slide was permeabilized with 0.5% Triton X-100 for 10 minutes, followed by blocking with 5% serum for 2 hours. The major antibodies used for immunofluorescence staining included inducible nitric oxide synthase (iNOS) (a marker for M1 macrophages, dilution 1:100, Thermo Fisher Scientific) and CD206 (a marker for M2 macrophages, dilution 1:100, Thermo Fisher Scientific). After incubation with the major antibodies at 37°C, the slides were washed with PBS. Then, fluorescently labeled secondary antibodies were added and incubated at room temperature for 1 hour. After a final PBS wash, the fluorescent labeling was observed under a fluorescence microscope.

[0036] Assessment of Ferraphobia-Related Indicators: Following specified transfection and treatment, supernatants from major cultured podocytes were collected. The degree of lipid peroxidation in cells was assessed by fluorescent staining using a BODIPY™ 581 / 591 C11 lipid peroxidation sensor (Thermo Fisher) according to the manufacturer's instructions. Malondialdehyde (MDA) levels were measured using an enzyme-linked immunosorbent assay (ELISA) kit (Abcam) according to the manufacturer's instructions. Glutathione (GSH) activity was measured using a total GSH assay kit (Beyotime). Intracellular iron content in lysates of major cultured podocytes was detected using an iron assay kit (Sigma-Aldrich).

[0037] Example 1: Abnormal expression of the CYBB gene, associated with ferroptosis, was observed in patients with lupus nephritis. To identify biomarkers for lupus nephritis (LN), this embodiment analyzed the GSE72326 dataset of transcriptomic data from healthy individuals and LN patients. Figure 1 Bioinformatics mining of differentially expressed genes in patients with lupus nephritis Figure 1 A is a hierarchical clustering heatmap of gene expression profiles of differentially expressed genes between healthy controls and systemic lupus erythematosus (SLE), with |LogFC|≥0 and p-value<0.05 for differentially expressed genes; Figure 1 B is a volcano plot of the fold change in gene expression (LogFC) between the two groups and the p-value obtained from the significance test, showing a significant difference; Figure 1C represents the enrichment analysis of DEGs based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO). GO functions include biological processes (BP), cellular components (CC), and molecular functions (MF). DEG analysis showed significant enrichment of immune-related biological processes, which is consistent with the expectation that SLE is an autoimmune disease. In addition, components related to cell vacuolar structure were also enriched, suggesting the involvement of programmed cell death pathways such as autophagy and ferroptosis. Figure 1 D represents the intersection of differentially expressed genes (DEGs) in lupus nephritis, macrophage polarization genes, and ferroptosis genes. When DEGs are cross-referenced with macrophage polarization-related genes, 9 genes are identified. When DEGs are cross-referenced with ferroptosis-related genes, 55 genes are identified. Figure 1 E represents the protein-protein interaction (PPI) network of macrophage polarization genes and SLE-related ferroptosis genes; Figure 1 F represents the gene with the most close interaction among SLE-related macrophage polarization genes and ferroptosis genes, identified from the GSE72326 dataset using a Sankey diagram; Figure 1 G shows the expression of the CYBB gene under normal conditions and in systemic lupus erythematosus (SLE), and found that the CYBB gene is significantly upregulated in the SLE group; Figure 1 H represents a correlation heatmap of metabolic differentials. The color intensity indicates the strength of the correlation, with blue indicating a positive correlation and red indicating a negative correlation. The heatmap shows that CYBB is significantly positively correlated with ferroptosis and pro-inflammatory M1 macrophage phenotype in LN.

[0038] Example 2: Upregulation of CYBB expression in hypoxia / reoxygenation-induced podocyte ferroptosis In this embodiment, primary podocytes were isolated from the kidneys of 8-10 week old MRL / MpJ mice, and a hypoxia / reoxygenation (H / R) model was established. Figure 2 A represents the analysis of primary podocytes from MRL / MpJ mice treated with hypoxia / reoxygenation (H / R) and Fer-1 (ferroptosis inhibitor ferrostatin-1) for 72 hours, as well as the control group, using continuous CCK-8 assays. Figure 2 B. To assess the number of viable cells, CFDA-SE staining was performed on the above cells. Figure 2 As shown in A and 2B, when podocytes are treated with H / R, cell viability decreases significantly, while the application of the ferroptosis inhibitor ferrostatin-1 (Fer-1) can alleviate this loss of viability. Figure 2The C11 probe, designated BODIPY 581 / 591, indicates the lipid peroxidation level in H / R-treated podocytes with and without Fer-1. The probe exhibits red fluorescence in its basal state and transforms into green fluorescence upon oxidation by reactive oxygen species (ROS). The green fluorescence serves as an indicator of ROS levels. Figure 2 D represents the malondialdehyde (MDA) level in podocytes after H / R and Fer-1 treatment, from... Figure 2 In C and 2D, it was observed that reactive oxygen species (ROS) and the lipid peroxidation marker malondialdehyde (MDA) significantly increased after H / R treatment, which corresponds to a decrease in cell viability. ROS and MDA significantly decreased after Fer-1 treatment. Figure 2 E represents the Western blot analysis showing the expression levels of CYBB, ferroptosis-related protein ACSL4, and ferroptosis negative regulator protein GPX4 in podocytes after H / R and Fer-1 treatment. The figure shows that ACSL4 significantly increased and GPX4 significantly decreased. Figure 2 F shows the mitochondrial morphology of podocytes after H / R and Fer-1 treatment. The arrows indicate mitochondria with cristae damage, indicating that under H / R treatment, podocytes exhibit the disappearance of mitochondrial cristae and rupture of the outer membrane, which are subcellular characteristics of ferroptosis. The application of the ferroptosis inhibitor Fer-1 improved the damage to mitochondrial cristae.

[0039] The molecular and imaging evidence presented in this embodiment indicates that podocytes undergo ferroptosis under H / R treatment, leading to decreased cell viability. Furthermore, CYBB expression levels are positively correlated with increased ferroptosis and decrease upon inhibition of ferroptosis.

[0040] Ferric death exists in the LN model in Example 3. MRL-lpr / lpr (MRL / lpr) LN mice exhibit abnormal proliferation of T and B cells, leading to systemic autoimmunity and SLE phenotype. In this example, primary podocytes were isolated from the kidneys of MRL / lpr mice, and primary podocytes from MRL / MpJ mice were used as a control to establish a cellular LN model.

[0041] from Figure 3 It was observed that the kidneys of MRL / lpr mice exhibited pathological characteristics of LN. Figure 3A shows the HE staining and MASSON staining results of kidney tissue sections from the control group (MRL / MpJ) and the LN group (MRL / lpr). As can be seen from the figure, the kidney sections of MRL / lpr mice show mild enlargement of glomeruli, open capillary lumen, diffuse thickening of glomerular basement membrane, and vacuolar degeneration of basement membrane. These features are consistent with the pathology of nephritis. Figure 3 B represents the ratio of kidney to body weight in the two groups of mice. The increased ratio of kidney to body weight in LN mice indicates kidney swelling. Figure 3 C Figure 3 Biochemical analysis of E showed increased levels of BUN and serum creatinine, accompanied by elevated levels of renal tissue inflammatory factors IL-1β, TNF-α, and IL-6; Figure 3 D represents the levels of ferroptosis markers MDA, iron ions, and glutamine in kidney tissue. The figure shows that MDA and iron ion levels increase while GSH (the negative regulator of ferroptosis) levels decrease, indicating the presence of ferroptosis in this LN model.

[0042] Example 4: CYBB participates in regulating ferroptosis in LN podocytes In this embodiment, the expression of CYBB in LN model cells was knocked down using the sh-CYBB plasmid. Figure 4 A represents the changes in primary podocytes from MRL / MpJ (control) and MRL / lpr (LN) mice over 72 hours with or without CYBB knockdown (sh-CYBB) as determined by continuous CCK-8 assay. Figure 4 B represents the number of surviving cells assessed by a single CFDA-SE staining. CCK-8 assay and CFDA staining showed that sh-CYBB treatment significantly improved the viability and survival rate of LN cells, while the viability of LN model cells was significantly lower than that of control cells. Figure 4 C is a transmission electron microscope (TEM) image showing the mitochondrial morphology of podocytes from MRL / MpJ (control) and MRL / lpr (LN) mice. The TEM image shows that primary cultured LN cells exhibit smaller mitochondria, increased membrane density, and darker color, which are consistent with ferroptosis. In sh-CYBB treated LN cells, the mitochondria are lighter in color compared to untreated LN cells. Figure 4 D represents the expression levels of CYBB, ferroptosis-associated protein ACSL4, and ferroptosis-negative regulatory protein GPX4 in LN podocytes and podocytes transfected with shCYBB plasmid, as shown by Western blot analysis. Figure 4 E represents the ROS level, where the green fluorescence displayed by the fluorescent probe indicates the ROS level. Figure 4 F represents the level of GSH (negative regulator of ferroptosis) in LN podocytes and podocytes transfected with shCYBB plasmid. Figure 4 G represents LN podocytes and Fe in podocytes transfected with shCYBB plasmid.2+ (Levels related to ferrodeath) Figure 4 H represents the level of MDA (associated with ferroptosis) in LN podocytes and podocytes transfected with the shCYBB plasmid. The figure shows that at the molecular level, CYBB is naturally highly expressed in LN cells compared to the control group, while CYBB expression is reduced in LN+shCYBB cells compared to the LN group, indicating that the shCYBB plasmid was successfully transfected into LN podocytes and subsequently downregulated CYBB expression. As markers of ferroptosis, ACSL4, ROS, and MDA initially increased and then decreased, while GPX4 and GSH initially decreased and then rebounded, indicating that the upregulation of CYBB expression in LN model podocytes is accompanied by an increase in ferroptosis. Knockdown of CYBB can partially reverse this increase, indicating that although CYBB promotes ferroptosis, it is not a necessary factor for ferroptosis to occur.

[0043] Example 5: CYBB indirectly exacerbates ferroptosis in LN cells by inducing M1 polarization in macrophages. From Example 1 Figure 1 Bioinformatics analysis of H showed a strong positive correlation between CYBB, M1 macrophage polarization and ferroptosis. In this embodiment, primary podocytes (insertion chamber) and primary isolated BMDM cells (lower chamber) were co-cultured in the Transwell co-culture system. Figure 5 A is a schematic diagram illustrating the co-culture of primary podocytes isolated from MRL-lpr / lpr (MRL / lpr) (LN) or MRL / MpJ (control) mice with primary bone marrow-derived macrophages (BMDMs) from normal mice using a bilayer culture system. During the culture process, in this example, LPS and IL-4 were used to induce the primary isolated BMDM cells to polarize into pro-inflammatory M1 and anti-inflammatory M2 macrophages, respectively. Figure 5 B shows the immunofluorescence staining of macrophages. The figure shows the polarization type of macrophages co-cultured with healthy controls, LN and CYBB knockdown LN podocytes. iNOS staining indicates M1 macrophages, while CD206 staining indicates M2 macrophages. Figure 5 C represents the expression levels of polarization markers iNOS (M1) and CD206 (M2) in macrophages co-cultured with LN and CYBB knockdown LN podocytes after LPS (M1) and IL-4 (M2) induced polarization. Figure 5 As shown in B, M1 macrophages were sparse in the control group. However, when co-cultured with LN podocytes, M1 macrophage polarization increased significantly. Conversely, when co-cultured with CYBB-knockdown LN podocytes, M1 polarization decreased significantly, while M2 polarization increased. Western blot analysis of iNOS and CD206 in macrophages confirmed these findings. Figure 5D shows the expression levels of CYBB and ferroptosis-related markers ACSL4 and GPX4 in LN and CYBB-knockdown podocytes co-cultured with polarized macrophages (as described in C). Western blot results of podocyte lysates showed that CYBB expression was reduced in the shCYBB transfection group compared with its respective control group, confirming the success of CYBB knockdown regardless of whether LPS or IL-4 was used. Figure 5 E represents the cell viability of LN podocytes co-cultured with LPS-induced polarized macrophages before and after CYBB knockdown, as shown by continuous CCK-8 assays. Figure 5 F shows the cell viability of LN podocytes co-cultured with IL-4-induced polarized macrophages before and after CYBB knockdown, as determined by continuous CCK-8 assays. CCK-8 staining in both figures shows that after CYBB knockdown, podocytes increased more rapidly and reached a higher endpoint value, indicating enhanced cell viability. Figure 5 G represents the MDA level in LN podocytes co-cultured with macrophages before and after CYBB knockdown. Figure 5 H represents the GSH level in podocytes. Figure 5 I represents the ROS level (green fluorescence) in podocytes, from... Figure 5 D and 5G-I showed decreased ACSL4 expression, reduced ROS level, decreased MDA level, while increased GPX4 and GSH expression. These results indicate that the increase in cell viability is due to reduced ferroptosis after CYBB knockdown. The comparison between LPS-NC and IL-4-NC further proves that LPS-induced M1 macrophage polarization increases ferroptosis in podocytes.

[0044] In summary, under the pathophysiological conditions of LN, CYBB is overexpressed in podocytes. This overexpression not only directly leads to ferroptosis in podocytes themselves, but also induces chemotaxis of monocytes to M1 macrophages in some way. These M1 macrophages, in turn, further exacerbate ferroptosis in podocytes. M1 macrophages are sparse in the control group, but when co-cultured with LN podocytes, M1 macrophage polarization is significantly increased.

[0045] Example 6: CYBB induces macrophage M1 polarization by promoting ferroptosis and potential intercellular communication mechanisms. This embodiment investigates how CYBB affects the polarization type of monocytes. Erastin (a ferroptosis inducer) was added to a co-culture system of LN cells and macrophages to induce ferroptosis. Figure 6A shows the expression levels of polarization markers iNOS (M1) and CD206 (M2) in macrophages co-cultured with podocytes with or without CYBB knockdown, as determined by Western blot analysis. The results showed that M1 polarization was significantly reduced and M2 polarization was significantly increased in macrophages co-cultured with CYBB knockdown podocytes under Erastin treatment. Figure 6 B represents the expression levels of CYBB, ACSL4, and GPX4 in podocytes transfected with shNC (negative control) or shCYBB plasmid and co-cultured with macrophages, as shown by Western blot analysis. Figure 6 C represents the cell viability of podocytes transfected with shNC or shCYBB plasmids and co-cultured with macrophages, as shown by continuous CCK-8 assays. Figure 6 D represents the ROS level in podocytes transfected with shNC or shCYBB plasmids and co-cultured with macrophages. Figure 6 E represents the MDA level in podocytes transfected with shNC or shCYBB plasmids and co-cultured with macrophages. Figure 6 F represents Fe in podocytes transfected with shNC or shCYBB plasmids and co-cultured with macrophages. 2+ level, Figure 6 G represents the GSH level in podocytes transfected with shNC or shCYBB plasmids and co-cultured with macrophages. For podocytes, CCK-8 assays showed that CYBB knockdown maintained cell viability under Erastin-induced ferroptosis. Simultaneously, ACSL4 expression was reduced, ROS levels decreased, MDA levels decreased, and Fe... 2+ Reduced CYBB levels and decreased GSH levels, along with increased GPX4 expression, indicate that knocking down CYBB in LN cells to suppress its high expression affects the efficiency of Erastin-induced ferroptosis, thereby reducing macrophage M1 polarization.

[0046] Besides ferroptosis leading to leakage of cell contents and dead cell debris recruiting macrophages, cell communication also exists between tissue cells and macrophages, such as the CCL2-CCR2 axis: under certain conditions, tissue cells actively release chemokines such as CCL2, which bind to the CCL2 receptor on macrophages, causing M1 polarization of surrounding macrophages. Example 1 identified the CCL2-CCR2 gene pair in previous bioinformatics analysis (…). Figure 1 Therefore, qPCR was performed on LN cells and macrophages to examine the transcriptional levels of CCL2 and CCR2. Figure 6 H represents the mRNA transcription levels of CCL2 and CCR2 in podocytes transfected with shNC or shCYBB plasmids and co-cultured with macrophages. Figure 6I represents the association between CYBB and SRC proteins shown in the STRING database. Figure 6 J represents the expression level of SRC in podocytes with CYBB knockdown under Erastin-induced ferroptosis conditions, as shown by Western blot analysis. The results showed that CYBB knockdown led to a decrease in the transcriptional levels of CCL2 and CCR2, consistent with expectations. The transcriptional levels of CCL2 and CCR2 were positively correlated with CYBB and also with the degree of M1 polarization. This suggests the involvement of intercellular communication mechanisms in the process of macrophage M1 polarization induced by high CYBB expression in LN cells. In addition to the CCL2-CCR2 axis, Example 1, based on bioinformatics analysis, identified genes related to CYBB-SRC. We found that besides the high expression of SRC and CYBB in SLE patients, SRC expression was also positively correlated with CYBB expression under Erastin-induced ferroptosis.

[0047] Example 7: SRC and CYBB interact directly in podocytes, synergistically mediating ferroptosis. This embodiment uses bioinformatics indicators and expression correlations to conduct co-immunoprecipitation (Co-IP) and fluorescence co-localization experiments to directly explore the interaction between CYBB and SRC. Figure 7 A. Immunoprecipitation (Co-IP) was performed using LN podocyte lysates. Agarose beads conjugated with anti-SRC antibodies were used to immunoprecipitate the proteins, followed by Western blotting (IB) analysis with anti-CYBB and anti-SRC antibodies. Agarose beads conjugated with homologous nonspecific IgG were used as a negative control. As a positive control, Western blotting analysis was performed directly on the lysates using anti-CYBB and anti-SRC antibodies to assess the levels of intrinsic CYBB and SRC in these LN podocytes. Figure 7 B represents the immunoprecipitation of CYBB-containing protein complexes from LN podocyte lysates using agarose beads conjugated with anti-IgG and anti-CYBB antibodies, followed by Western blotting analysis of the eluted proteins using anti-SRC and anti-CYBB antibodies. Figure 7 C shows the colocalization of SRC (red) and CYBB (green) in LN podocytes using immunofluorescence staining. The cell nuclei are stained with DAPI (blue). As can be seen from the above figure, the Co-IP results show that CYBB antibody can precipitate SRC, and SRC antibody can precipitate CYBB. Immunofluorescence staining shows the colocalization of SRC and CYBB in podocytes. Figure 7 D, a CCK-8 assay, showed the cell viability of LN podocytes with SRC knockdown or CYBB overexpression under Erastin-induced ferroptosis. Figure 7 E represents the malondialdehyde (MDA) level in LN podocytes with SRC knockdown or CYBB overexpression under Erastin-induced ferroptosis. Figure 7F represents the glutathione (GSH) level in LN podocytes with SRC knockdown or CYBB overexpression under Erastin-induced ferroptosis. Figure 7 G represents Fe in LN podocytes with SRC knockdown or CYBB overexpression under Erastin-induced ferroptosis. 2+ The above experiments on SRC and CYBB knockdown and overexpression showed that, under Erastin-induced ferroptosis, CYBB overexpression significantly promoted ferroptosis, while SRC knockdown alleviated the enhanced ferroptosis caused by CYBB overexpression. Furthermore, even under normal CYBB expression, SRC knockdown increased podocyte viability by reducing Erastin-induced ferroptosis. These findings indicate that SRC and CYBB play a promoting role in podocyte ferroptosis; SRC knockdown mitigates the effect of CYBB overexpression, and vice versa, CYBB overexpression compensates for the effect of SRC knockdown. This suggests that SRC and CYBB have a synergistic rather than competitive relationship in promoting ferroptosis.

[0048] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered within the scope of the claims and specification of the present invention.

Claims

1. Application of reagents for detecting SRC expression in the preparation of products for the diagnosis and treatment of lupus nephritis.

2. The application according to claim 1, characterized in that, The SRC is highly expressed in patients with lupus nephritis.

3. The application according to claim 1, characterized in that, The product includes SRC detection reagents.

4. The application according to claim 3, characterized in that, The detection reagents include those used to detect SRC expression levels via quantitative real-time PCR.

5. Application of reagents for detecting CYBB expression in the preparation of products for the diagnosis and treatment of lupus nephritis.

6. The application according to claim 5, characterized in that, The CYBB is highly expressed in patients with lupus nephritis.

7. The application according to claim 5, characterized in that, The product includes CYBB detection reagents.

8. The application according to claim 7, characterized in that, The detection reagents include those used to detect CYBB expression levels by quantitative real-time PCR.

9. Application of reagents for detecting SRC and CYBB expression in the preparation of products for the diagnosis and treatment of lupus nephritis.

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