Gout biomarker HIF-1alpha and application thereof

Abstract

The invention relates to the field of biological medicine, in particular to application of a reagent for measuring or regulating the expression level of HIF-1alpha in preparation of a kit for diagnosing or gout, the kit containing the reagent and a gout prediction device. The HIF-1alpha is applied as a gout diagnosis target and a treatment target for the first time, the polarization degree of Th17 cells in an inflammatory microenvironment of a gout patient can be specifically reflected, early dynamic monitoring of gout related inflammatory activities is achieved, polarization of Th17 cells and expression of related proinflammatory factors are inhibited in a targeted mode, and a new strategy is provided for prediction, diagnosis and treatment of gout.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the use of reagents for measuring or regulating HIF-1α expression levels in the preparation of kits for diagnosing or treating gout, kits containing the above-mentioned reagents, and a gout prediction device. Background Technology

[0002] Gout is a chronic inflammatory joint disease caused by hyperuricemia and sodium urate crystal deposition. Its global prevalence in adults has reached 0.68%-3.90%. The accompanying joint dysfunction, metabolic syndrome, and multi-system complications significantly reduce patients' quality of life. Although some progress has been made in recent years in treatment with anti-inflammatory drugs, uric acid-lowering therapies, and anti-IL-1β biologics, their severe side effects, high recurrence rates, and the high cost of biologics highlight the urgent need to find safe, effective, and affordable treatments for gout.

[0003] Current clinical interventions for gout primarily rely on xanthine oxidase inhibitors (such as allopurinol) to control serum uric acid levels. However, this strategy only targets the upstream cause of hyperuricemia and lacks direct regulatory effects on the inflammatory cascade triggered by existing crystal deposition. Furthermore, related diagnostic techniques are mostly based on uric acid testing and imaging, making it difficult to achieve dynamic monitoring of the early inflammatory process. Although T cell dysfunction, especially hyperactivity of helper T cells 17 (Th17), plays a fundamental role in the development and progression of gout, the key molecular targets regulating it remain unclear, resulting in slow progress in the development of precision diagnostic tools and targeted therapies.

[0004] Recent studies have shown that HIF-1α, as a core regulator of immune metabolic reprogramming, has played an important role in various inflammatory diseases. However, recent research indicates that HIF-1α inhibits SOCS3. Upregulation of SOCS3 plays a role in suppressing inflammation and alleviating gout pain, while reduced SOCS3 can exacerbate gout inflammation (Wan, Jun et al. "SOCS3 blocks HIF-1α expression to inhibit proliferation and angiogenesis of human small cell lung cancer by downregulating activation of Akt, but not STAT3." Molecular Medicine Reports vol. 12, 1(2015): 83-92. doi:10.3892 / mmr.2015.3368; Fan, Wen et al. "Ozone alleviates MSU-induced acute gout pain via upregulating AMPK / GAS6 / MerTK / SOCS3 signaling pathway." Journal of Translational Medicine vol. 21, 1). (890.8Dec.2023, doi:10.1186 / s12967-023-04769-1), therefore its role in gout has not yet been elucidated.

[0005] There is an urgent need in this field to discover molecular markers that can specifically regulate Th17 cell polarization, in order to fill the gap in the lack of biomarkers for the early diagnosis of gout and to provide a new pathway for the development of targeted anti-inflammatory therapies. Summary of the Invention

[0006] The present invention aims to at least partially solve one of the technical problems in the related art.

[0007] Therefore, an embodiment of the first aspect of the present invention proposes the use of a reagent for determining the expression level of HIF-1α in the preparation of a kit for diagnosing gout, wherein the reagent is used to determine the mRNA or protein content of HIF-1α in a biological sample. This first application of HIF-1α as a diagnostic target for gout can specifically reflect the polarization degree of Th17 cells in the inflammatory microenvironment of gout patients, breaking through the traditional diagnostic reliance on a single blood uric acid index, enabling early dynamic monitoring of gout-related inflammatory activity, and the detection strategy based on non-invasive samples such as blood is beneficial for clinical application.

[0008] In some embodiments, the biological sample is blood, serum, colon tissue, spleen tissue, peripheral blood mononuclear cells, macrophages, CD4+. + T cells, CD8 + T cells and B cells.

[0009] In some embodiments, the biological sample is CD4. + T cells.

[0010] The second aspect of this invention discloses the use of a reagent for regulating HIF-1α expression levels in the preparation of a drug for treating gout. This is the first application of HIF-1α as a therapeutic target for gout. By specifically upregulating HIF-1α expression, it targets and inhibits excessive polarization of Th17 cells and the expression of related pro-inflammatory factors, thus blocking the progression of gouty arthritis from the core of inflammation. Compared to traditional uric acid-lowering drugs, its intervention in the Th17 cell-driven inflammatory cascade can significantly alleviate symptoms such as joint redness, swelling, heat, and pain, and reduce tissue damage induced by sodium urate crystal deposition, providing a new strategy for gout treatment.

[0011] In some embodiments, the reagent that regulates HIF-1α expression levels treats Th17 cells by regulating their polarization.

[0012] In some embodiments, the reagent that regulates HIF-1α expression levels reduces Th17 cell polarization by decreasing the expression of the HIF-1α gene.

[0013] In some embodiments, treating gout includes improving at least one of the following gout-related indicators:

[0014] Lower uric acid levels;

[0015] Reduce inflammatory cell infiltration in the footpads;

[0016] Relieve swelling;

[0017] Increase pain threshold;

[0018] Reduce serum inflammatory infiltration; and

[0019] Reduce IL-17A expression.

[0020] In some embodiments, treating gout includes improving gout-related indicators in a subject, wherein the subject is a mammal, preferably a mouse and / or a human.

[0021] In some embodiments, the reagents for regulating HIF-1α levels include diosgenin and PX-478.

[0022] A third aspect of the present invention provides a kit comprising reagents for determining HIF-1α expression levels and / or reagents for regulating HIF-1α expression levels.

[0023] A fourth aspect of the present invention provides a gout prediction device, characterized in that the device comprises:

[0024] A sample collection unit, the sample collection unit being adapted to collect biological samples from a subject;

[0025] A determining unit is used to determine the expression level of the HIF-1α gene in the obtained biological sample;

[0026] The prediction unit compares the HIF-1α expression level with a reference value.

[0027] The advantages and technical effects brought about by the independent claims according to the embodiments of the present invention are as follows:

[0028] (1) This invention uses HIF-1α as a specific diagnostic marker for gout for the first time. By detecting its mRNA / protein level, it indirectly quantifies the degree of Th17 polarization, breaking through the limitations of traditional lagging diagnosis that relies on blood uric acid or imaging. It enables early dynamic monitoring of inflammatory activity (such as the asymptomatic hyperuricemia stage) and is significantly beneficial for clinical promotion based on non-invasive methods such as blood.

[0029] (2) This invention is the first to use HIF-1α as the intervention target. By downregulating its expression, it specifically inhibits Th17 cell polarization and the release of pro-inflammatory factors, and intervenes in the inflammatory cascade response driven by Th17 cells. This can significantly improve gout-related indicators and provide a new strategy for gout treatment.

[0030] (3) The present invention provides for the first time a kit containing HIF-1α detection / regulation reagents, which can be combined with multiple indicators (such as HIF-1α expression level and inflammatory factor level) for gout diagnosis, improving diagnostic accuracy and providing a real-time efficacy evaluation tool for targeted therapy. Attached Figure Description

[0031] Figure 1 This is a bar chart illustrating the proportion of immune cells, showing the proportion of immune cell populations in patients with hyperuricemia and normal healthy individuals according to embodiments of the present invention, where HC represents healthy individuals and HUA represents patients with hyperuricemia.

[0032] Figure 2This is a schematic diagram of differential gene pathway analysis, which shows the A. KEGG pathway enrichment analysis of differential genes between hyperuricemia patients and normal healthy individuals according to an embodiment of the present invention, which shows the metabolic-related pathways enriched in HUA patients; B. GSEA analysis map, which shows the metabolic pathways enriched in the HUA and HC groups, where HC represents healthy individuals and HUA represents hyperuricemia patients.

[0033] Figure 3 These are juvenile normal mice and hyperuricemia model mice according to embodiments of the present invention. CD4 + A bar chart showing the intracellular and extracellular uric acid levels in T cells.

[0034] Figure 4 These are normal mice and hyperuricemia model mice according to embodiments of the present invention. CD4 + A bar chart showing the intracellular and extracellular uric acid levels in T cell-induced Th1, Th2, Th17, and Treg cells. The chart illustrates: A. Changes in intracellular and extracellular uric acid levels in Th1 cells; B. Changes in intracellular and extracellular uric acid levels in Th2 cells; C. Changes in intracellular and extracellular uric acid levels in Th17 cells; D. Changes in intracellular and extracellular uric acid levels in Treg cells. The groups are as follows: WT normal mice CD4+ T cells; WT Th1: Th1 cells induced in normal mice; WT Th2: Th2 cells induced in normal mice; WT Th17: Th17 cells induced in normal mice; WT Treg: Treg cells induced in normal mice; KO Uox-KO mice CD4 + T cells; KO Th1: Th1 cells induced by Uox-KO mice; KO Th2: Th2 cells induced by Uox-KO mice; KO Th17: Th17 cells induced by Uox-KO mice; KO Treg: Treg cells induced by Uox-KO mice. No significant difference was observed in ns, *P<0.05, **P<0.01.

[0035] Figure 5 It is a CD4 according to an embodiment of the present invention. + A schematic diagram of HIF-1α gene and protein expression in T cells, showing AB. CD4 isolated from peripheral blood of gout patients and healthy controls (HCs). + qPCR and Western blot results of T cells, including CD4 in gout patients + The levels of HIF-1α mRNA and protein in T cells were significantly increased; CD4 was isolated from the peripheral blood of CD.Uox-KO mice and normal mice.+ qPCR and Western blot results of T cells, including CD4 in Uox-KO mice. + HIF-1α expression is upregulated in T cells.

[0036] Figure 6 This is a schematic diagram showing the measurement results of gout-related indicators after HIF-1α inhibition treatment in a mouse model of gout according to an embodiment of the present invention. It shows the serum uric acid (S-UA) and urinary uric acid (U-UA) levels in Uox-KO mice treated with AB.PX-478 (n=7-8); representative H&E staining images and pathological analysis of mouse paw pad tissue after C.PX-478 treatment (scale bar: 500μm); changes in paw pad swelling index and mechanical pain threshold in the DE.PX-478 treatment group; changes in renal function parameters after FG.PX-478 treatment; representative photographs and H&E analysis of kidney tissue in the H.PX-478 treatment group (scale bar: 250μm); serum and urinary uric acid levels in CD4cre HIF-1α fl / fl mice stimulated by a high-fat diet / MSU (n=7-8); K Representative images and H&E analysis of footpad tissue from CD4cre HIF-1αfl / fl mice (scale bar: 500 μm); changes in footpad swelling and pain threshold in LM.CD4cre HIF-1αfl / fl mice; representative photographs and H&E analysis of kidney tissue from N.CD4cre HIF-1αfl / fl mice (scale bar: 250 μm); renal function indicators in O.CD4cre HIF-1αfl / fl mice.

[0037] Figure 7 This is a schematic diagram of differential gene pathway analysis, showing the pathway analysis results of differential genes between gout patients and normal healthy individuals, gout model mice and normal mice according to embodiments of the present invention. It shows the CD4+ of A.CD4creHIF-1αfl / fl mice. + RNA sequencing analysis of T cells showed that HIF-1α deficiency led to the upregulation of 684 genes and the downregulation of 291 genes; B. KEGG pathway enrichment analysis showed that Th17 cell differentiation and IL-17 signaling pathways were inhibited in HIF-1α-deficient cells; CD. Gene set enrichment analysis showed that HIF-1α deficiency resulted in negative enrichment of genes related to Th17 differentiation and IL-17 signaling; E. CD4 in gout patients and healthy controls. + T cell RNA sequencing results; F. CD4 in gout patients + Volcano plot of differentially expressed genes on T cells (fold change >2.0 or <0.5); KEGG enrichment analysis of HIF-1 signaling pathway, Th17 cell differentiation and IL-17 signaling pathway in G. gout patients.

[0038] Figure 8 This is a schematic diagram of the Th17 cell level and inflammation level in each treatment group according to embodiments of the present invention, showing: A. Analysis of the proportion and absolute number of Th17 cells in peripheral blood of healthy controls (n=15) and gout patients (n=20); B. Distribution characteristics of Th17 cell populations in peripheral blood, intestine and spleen of Uox-KO mice (n=10); C. CD4+ in footpad tissue of Uox-KO mice. + (Red) IL17A + (Yellow) Statistical graph of immunofluorescence of double-positive cells (n=3); D. Changes in the proportion and number of Th17 cells in CD4cre HIF-1αfl / fl mice after high-fat diet / MSU stimulation; E. Dynamic changes in the Th17 cell population in PX-478-treated mice; FG. CD4+ cells in the footpad tissue of CD4cre HIF-1αfl / fl mice and PX-478-treated mice. + / IL17A + Cellular immunofluorescence contrast and its statistical graph (scale bar: 50 μm, n=3).

[0039] Figure 9 This is a schematic diagram of the Th17 cell level and inflammation level of each treatment group according to an embodiment of the present invention, showing A. the blocking effect of HIF-1α inhibition on the expansion of Th17 cells in vitro; B. comparison of serum IL-17A levels in each group (n=5-8). Detailed Implementation

[0040] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0041] This invention is based on the inventor's discoveries and understanding of the following facts and problems:

[0042] Embodiments of the present invention provide the use of a reagent for measuring or regulating HIF-1α expression levels in the preparation of a kit for diagnosing or treating gout, a kit comprising the above-mentioned reagent, and a gout prediction device.

[0043] An embodiment of the first aspect of this invention discloses the use of a reagent for determining HIF-1α expression levels in the preparation of a kit for diagnosing gout, wherein the reagent is used to determine the mRNA or protein content of HIF-1α in a biological sample. This first-time application of HIF-1α as a diagnostic target for gout specifically reflects the polarization degree of Th17 cells in the inflammatory microenvironment of gout patients, breaking through the traditional diagnostic reliance on a single blood uric acid index, enabling early dynamic monitoring of gout-related inflammatory activity, and the detection strategy based on non-invasive samples such as blood is beneficial for clinical application.

[0044] In some embodiments, the reagent is used to determine the mRNA content of HIF-1α in a biological sample.

[0045] In some embodiments, the biological sample is blood, serum, colon tissue, spleen tissue, peripheral blood mononuclear cells, macrophages, CD4+. + T cells, CD8 + T cells and B cells.

[0046] In some embodiments, the biological sample is CD4. + T cells.

[0047] The second aspect of this invention discloses the use of a reagent for regulating HIF-1α expression levels in the preparation of a drug for treating gout. This is the first application of HIF-1α as a therapeutic target for gout. By specifically upregulating HIF-1α expression, it targets and inhibits excessive polarization of Th17 cells and the expression of related pro-inflammatory factors, thus blocking the progression of gouty arthritis from the core of inflammation. Compared to traditional uric acid-lowering drugs, its intervention in the Th17 cell-driven inflammatory cascade can significantly alleviate symptoms such as joint redness, swelling, heat, and pain, and reduce tissue damage induced by sodium urate crystal deposition, providing a new strategy for gout treatment.

[0048] In some embodiments, the reagent that regulates HIF-1α expression levels treats Th17 cells by regulating their polarization.

[0049] In some embodiments, the reagent that regulates HIF-1α expression levels reduces Th17 cell polarization by decreasing the expression of the HIF-1α gene.

[0050] In some embodiments, treating the gout includes improving at least one of the following gout-related indicators: lowering uric acid levels; reducing inflammatory cell infiltration in the foot pads; relieving swelling; increasing the pain threshold; reducing serum inflammatory infiltration; and lowering IL-17A expression.

[0051] In some embodiments, treating gout includes improving gout-related indicators in a subject, wherein the subject is a mammal, preferably a mouse and / or a human.

[0052] In some embodiments, the reagents for regulating HIF-1α levels include diosgenin and PX-478. It is understood that the embodiments of the present invention are not intended to limit the reagents for regulating HIF-1α levels; any reagent capable of regulating HIF-1α levels to achieve the purpose of treating gout is covered within the scope of protection of the embodiments of the present invention.

[0053] A third aspect of the present invention provides a kit comprising reagents for determining HIF-1α expression levels and / or reagents for regulating HIF-1α expression levels.

[0054] A fourth aspect of the present invention provides a gout prediction device, characterized in that the device comprises: a sample collection unit adapted to collect biological samples from a subject; a determination unit configured to determine the expression level of the HIF-1α gene in the obtained biological samples; and a prediction unit comparing the HIF-1α expression level with a reference value.

[0055] In some embodiments, the risk of gout is predicted in the subjects based on the fact that their HIF-1α expression levels are lower than the reference value.

[0056] Example

[0057] Ethical Statement:

[0058] All animal experimental protocols were developed in accordance with the Guidelines for the Use and Care of Laboratory Animals and were reviewed by the Laboratory Animal Ethics Committee of Zhejiang University of Traditional Chinese Medicine (Approval No.: 20210600226, 2021), and comply with the relevant principles of the International Council for Laboratory Animal Science (ICLAS).

[0059] The collection of blood samples and related clinical data from 32 patients with gout and 25 healthy individuals was approved by the Ethics Committee of Zhejiang University of Traditional Chinese Medicine (Approval No.: 2023-KL-0825-1, 2023), and all participants signed informed consent forms. All experimental procedures involving human subjects complied with the ethical standards of the Declaration of Helsinki.

[0060] Blood samples and related clinical data from 32 patients with hyperuricemia and 25 healthy controls were collected from the First Affiliated Hospital, Second Affiliated Hospital, and Third Affiliated Hospital of Zhejiang University of Traditional Chinese Medicine (Approval No.: 20240329-6). All experimental procedures involving human subjects complied with the ethical standards of the Declaration of Helsinki.

[0061] Statistical analysis:

[0062] Data are expressed as mean ± standard error (SEM). Student's t-test (for normally distributed groups) or Mann-Whitney U test (for non-normally distributed groups) was used for comparisons between two groups; Kruskal-Wallis test or one-way ANOVA combined with Tukey multiple comparisons was used for comparisons among multiple groups. GraphPad Prism (GraphPad, USA) was used for analysis. Spearman's rank test was used to analyze the correlation between gout indicators and biomarkers. P < 0.05 was considered statistically significant.

[0063] Example 1: Identification of CD4+ immune cell subsets in human hyperuricemia + Significant changes in T cells and their related metabolic pathways, including the HIF-1 signaling pathway.

[0064] Hyperuricemia is a metabolic disease caused by purine metabolism disorder, and it is the most important underlying and direct cause of gout. In this embodiment, 10× single-cell RNA sequencing (10×scRNA-seq) was used to systematically analyze the peripheral blood immune cell profiles of hyperuricemia (HUA) patients and healthy controls (HC).

[0065] (1) Wash the sorted CD45-positive cells three times with 1×PBS. Each 1×10⁶ wash... 6 Cells were lysed using a pipette tip without RNase by adding 1 mL of Trizol reagent. After the cells were fully lysed, they were stored in a -80°C freezer.

[0066] (2) The construction and sequencing of single-cell libraries were outsourced to BGI Genomics. High-throughput sequencing was performed using the Illumina NovaSeq sequencer to obtain raw sequencing data.

[0067] (3) The raw data were preprocessed using CellRanger (v5.0.1) software to complete data quality control, deduplication and sequence alignment, and finally the gene expression matrix of each sample was generated.

[0068] (4) The gene expression matrix was then analyzed downstream using the R package Seurat (v3.2.0).

[0069] (5) Strict quality control of cells is performed based on the number of detected genes and the proportion of mitochondrial gene reads, low-quality cells and possible two-cell events are removed, and data normalization is completed.

[0070] (6) After data standardization, 2000 highly variable genes were screened from the entire dataset, and the cells were initially dimensionality reduced using principal component analysis (PCA).

[0071] (7) Nonlinear dimensionality reduction and cell clustering analysis were performed using the t-SNE algorithm to reveal the intrinsic relationships between different cell subpopulations.

[0072] (8) The FindAllMarkers function in the Seurat package (with the logFC threshold set to be greater than 0.25, minPct greater than 0.25, and Padj less than or equal to 0.05) was used to identify the characteristic marker genes of each cell cluster, and the cell types were annotated using the SCSA method.

[0073] (9) The FindMarkers function was used to screen differentially expressed genes among specific cell populations in detail.

[0074] (10) KEGG pathway enrichment and gene set enrichment analysis (GSEA) were performed on differentially expressed genes in patients with hyperuricemia compared with normal healthy individuals from single-cell sequencing data.

[0075] The results of the immune cell subset study showed that CD4+ was successfully identified in both groups of samples. + T cells, CD8 + Multiple immune cell subsets, including T cells, NK cells, B cells, monocytes, and dendritic cells. Figure 1 Among them, NK and CD4 + The percentage of T cells showed a decreasing trend; further statistical analysis clarified that CD4+ was present in HUA patients. + The absolute number and relative proportion of T cells were significantly reduced. These findings suggest that in hyperuricemia, the peripheral immune microenvironment undergoes significant remodeling, and CD4+... + T cells play a crucial role.

[0076] Further KEGG pathway enrichment and GSEA analysis revealed enrichment in purine metabolism, the HIF-1 signaling pathway, and glycolysis / gluconeogenesis, with these pathways significantly upregulated (see [link to KEGG pathway analysis]). Figure 2 ).

[0077] Example 2: CD4 was identified through a mouse immune cell subset study. + The effect of T cell-induced differentiation of Th17 cell subsets on uric acid levels

[0078] This embodiment measures... CD4 + Intracellular and extracellular uric acid levels of T cells and their induced differentiation subsets to further explore different CD4+ cells. + The regulatory role of T cell subsets in uric acid levels.

[0079] 2.1 Collection CD4 + T cells

[0080] Spleens were isolated from C57BL / 6 mice aged 4–6 weeks. CD4 + T cells were separated using the EasySep magnetic bead sorting kit. TM Mouse CD4 + The T Cell Isolation Kit (catalog number: 19765, STEMCELL) yields high-purity (>90%) cells. CD4 + T cells were analyzed by flow cytometry to detect the expression of cell markers CD44 (negative) and CD62L (positive) to verify differentiation effect and functional status.

[0081] 2.2 Induction of Differentiation

[0082] To study different CD4 +The function of T cell subsets was assessed using the following polarization conditions: For Th1 cell polarization, anti-CD3 (purchased from BD; catalog number 553057), anti-CD28 (purchased from BD; catalog number 553294), recombinant mouse IL-12 (30 ng / mL) (purchased from Bio; catalog number 577002), recombinant mouse IL-2 (5 ng / mL) (Bio; catalog number 575402), and anti-mouse IL-4 (10 μg / mL) (BD; catalog number 554432 or purchased from Bio; catalog number 504122) were added; for Th2 cell polarization, [the following was added]... Add anti-CD3 (purchased from BD; catalog number 553057), anti-CD28 (purchased from BD; catalog number 553294), recombinant mouse IL-4 (10 ng / mL) (purchased from Bio; catalog number 574302), recombinant mouse IL-2 (50 ng / mL) (purchased from Bio; catalog number 575402), and anti-mouse IFN-γ (10 μg / mL) (purchased from BD; catalog number 554408); when polarizing Th17 cells, add anti-CD3 (purchased from BD; catalog number 553057) and anti-CD28 (purchased from BD; catalog number 553294). Recombinant mouse IL-6 (100 ng / mL) (purchased from BD; catalog number 554587), recombinant mouse TGF-β1 (3 ng / mL) (purchased from Bio; catalog number 763104), recombinant mouse IL-1β (20 ng / mL) (purchased from Bio; catalog number 575102), recombinant mouse IL-23 (10 ng / mL) (purchased from Bio; catalog number 589002), anti-mouse IL-4 (10 μg / mL) (BD; catalog number 554432) and anti-mouse IFN-γ (10 μg / mL) ( ... IL-6 (100 ng / mL) (purchased from BD; catalog number 554587), recombinant mouse TGF-β1 (3 ng / mL) (purchased from Bio; catalog number 763104), recombinant mouse IL-1β (20 ng / mL) (purchased from Bio; catalog number 575102), recombinant mouse IL-23 (10 ng / mL) (purchased from Bio; catalog number 589002), anti-mouse IL-4 (10 μg / mL) (BD 4408); When polarizing Treg cells, anti-CD3 (purchased from BD; catalog number 553057), anti-CD28 (purchased from BD; catalog number 553294), recombinant mouse IL-2 (20 ng / mL) (purchased from Bio; catalog number 575402), recombinant mouse TGF-β1 (10 ng / mL) (purchased from Bio; catalog number 763104), anti-mouse IL-4 (10 μg / mL) (purchased from BD; catalog number 554432) and anti-mouse IFN-γ (10 μg / mL) (purchased from BD; catalog number 554408) were added. All cells were cultured at 37°C in a 5% CO2 incubator for 6 days, and the polarization effect was verified by flow cytometry.

[0083] 2.3 Measurement of uric acid levels

[0084] Cells were collected and centrifuged at 1500 rpm for 5 min to collect the supernatant (for measuring extracellular uric acid content). The cell pellet was washed three times with 1 mL PBS and then resuspended in 100 μL PBS for sonication on ice. The cells were then centrifuged at 1500 rpm for 5 min to collect the supernatant (for measuring intracellular uric acid content). Uric acid content was measured using the Nanjing Jiancheng Uric Acid Detection Kit (catalog number: C012-2-1) according to the kit instructions. The results showed that undifferentiated cells in normal mice and hyperuricemic model mice... CD4 + The levels of uric acid synthesized and secreted by T cells in vitro were low and showed no difference. Figure 3 However, after induced differentiation, Th1( Figure 4 (P<0.05) and Th17 ( Figure 4 (P<0.05, P<0.01) All cells showed significantly enhanced ability to synthesize and secrete uric acid, while the ability of induced differentiated Th2 and Treg cell subsets to produce uric acid remained unchanged, suggesting that CD4+ cells... + Th1 and Th17 cell subsets are the core groups that cause elevated uric acid levels in the body.

[0085] In conclusion, CD4 + The pro-inflammatory cell subsets resulting from abnormal T cell differentiation play a crucial role in the pathogenesis of hyperuricemia. The results showed that the model mice exhibited a significant increase in the proportion of Th1 and Th17 cell subsets. Further cell function experiments and adoptive immunotherapy experiments confirmed the role of CD4+. + T cell activation and differentiation can directly promote uric acid production in the body. The HIF-1 signaling pathway is a key pathway in this process. Given that hyperuricemia is a metabolic disease caused by purine metabolism disorder and is the most important underlying and direct cause of gout, it is necessary to investigate CD4+. + We studied the genes in the HIF-1 signaling pathway of T cells.

[0086] Example 3 CD4 + The role of HIF-1α in T cells in gout

[0087] 3.1 CD4 + Upregulation of HIF-1α in T cells is positively correlated with gout.

[0088] This embodiment investigates CD4 in gout patients and mouse models. + Expression of HIF-1α in T cells.

[0089] This embodiment isolated peripheral CD4 from gout patients. +T cells were analyzed to detect HIF-1α expression using qPCR and Western blot. Similarly, CD4+ expression was detected in the corresponding Uox-KO mice (a widely used model mouse for hyperuricemia and gout research) using qPCR and Western blot. + HIF-1α expression in T cells.

[0090] The results showed that, compared with healthy controls, gout patients had higher CD4 counts. + The levels of HIF-1α mRNA and protein in T cells were significantly elevated (see [link to relevant documentation]). Figure 5 (AB). Furthermore, consistent with human data, Uox-KO's CD4... + HIF-1α in T cells is significantly upregulated at both the transcriptional and translational levels. Figure 5 (CD). Correlation analysis showed that CD4 + The mRNA level of HIF-1α in T cells was significantly positively correlated with disease severity. These results indicate that CD4 + High expression of HIF-1α in T cells is an important driving factor in the pathogenesis of gout.

[0091] 3.2 Inhibition of CD4 + HIF-1α in T cells can alleviate gout.

[0092] This embodiment demonstrates the effect of inhibiting CD4 in a gout mouse model. + The expression of HIF-1α in T cells and the role of inflammation in gout.

[0093] In this embodiment, Uox-KO mice were intraperitoneally injected with 20 mg / kg of the HIF-1α inhibitor PX-478 (MCE, HY-10231) every two days, and gout-related indicators were measured. In addition, CD4+ was constructed. + T cell-specific HIF-1α conditional knockout mice (i.e., CD4cre HIF-1αfl / fl mice) were used, and gout-related markers were measured.

[0094] The results showed that, compared with the untreated group, PX-478 significantly reduced serum and urinary uric acid levels, while improving footpad inflammation, including a reduction in swelling and inflammatory cell infiltration. PX-478 also improved renal function, such as the restoration of serum and urinary creatinine levels, the restoration of urinary protein and albumin concentrations, and reduced renal tubular atrophy, glomerular lesions, and fibrosis (see [link to study]). Figure 6 (AH). This further supports the key role of HIF-1α in the progression of gout.

[0095] And CD4 +In T-cell-specific HIF-1α conditional knockout mice, related indicators were significantly alleviated, indicating that HIF-1α knockout can significantly reduce hyperuricemia and inflammatory response, and alleviate renal pathological damage. Figure 6 (IO). This reveals CD4 + The causal relationship between HIF-1α in T cells and gout progression further clarifies that it can serve as a therapeutic target.

[0096] 3.3 HIF-1α regulates CD4 in gout + Th17 cell proliferation

[0097] This embodiment investigates Th17 differentiation in a CD4cre HIF-1αfl / fl mouse model.

[0098] (1) In this embodiment, CD4c in the spleen of CD4c HIF-1αfl / fl mice was analyzed. + T cells underwent RNA sequencing, followed by routine transcriptome gene expression analysis, differential gene expression analysis, and KEGG enrichment analysis of differentially expressed genes. Furthermore, this embodiment analyzed CD4+ expression in gout patients. + The T cells were processed and analyzed in the same way as blood samples from healthy individuals.

[0099] (2) In this embodiment, the proportion of Th17 in the peripheral blood of gout patients and healthy controls was analyzed by flow cytometry. Similarly, the proportion of Th17 and IL-17A in the blood, intestine, spleen and footpad of Uox-KO mice, HIF-1α knockout mice and Uox-KO mice treated with PX-478 were analyzed.

[0100] Transcriptome analysis showed that HIF-1α knockout led to the upregulation of 684 genes and the downregulation of 291 genes. KEGG analysis indicated significant enrichment of Th17 differentiation and the IL-17 signaling pathway. GSEA analysis showed negative enrichment of both Th17-related genes and the IL-17 pathway, suggesting that HIF-1α is involved in regulating Th17 differentiation. In human samples, CD4+ in gout patients... + T cells showed significant differences in transcriptional characteristics compared to healthy individuals, with significant activation of the HIF-1 signaling pathway, as well as the Th17 and IL-17 pathways. Figure 7 ).

[0101] Flow cytometry confirmed a significantly increased proportion of Th17 cells in gout patients, and a similarly significant increase in the proportion of Th17 cells in the blood, intestines, spleen, and footpads of Uox-KO mice. However, HIF-1α knockout or PX-478 treatment significantly reduced the number of Th17 cells. In vitro experiments also showed that PX-478 inhibited Th17 differentiation, and IL-17A concentration decreased synchronously. Figure 8 and Figure 9 The results showed that HIF-1α promotes Th17 cell proliferation and IL-17 production, and is an important regulatory factor in the inflammatory pathology of gout.

[0102] In summary, this study establishes CD4 for the first time. + The mechanism by which HIF-1α in T cells promotes Th17 differentiation and drives gout has been proposed, leading to a new strategy of HIF-1α targeted therapy represented by HIF-1α inhibitors, which has advanced the understanding of gout as a disease of abnormal T cell metabolism.

[0103] In this invention, the terms "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to a specific feature, structure, material, or characteristic described in connection with that embodiment or example, which is included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0104] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. The use of a reagent for determining HIF-1α expression levels in the preparation of a kit for diagnosing gout. The reagents described herein are used to determine the mRNA or protein content of HIF-1α in biological samples.

2. The use according to claim 1, characterized in that, The biological samples included blood, serum, colon tissue, spleen tissue, peripheral blood mononuclear cells, macrophages, and CD4+. + T cells, CD8 + T cells, B cells. Preferably, the biological sample is CD4. + T cells.

3. Use of a reagent for regulating HIF-1α expression levels in the preparation of a drug for treating gout.

4. The use according to claim 3, characterized in that, The reagent that regulates HIF-1α expression levels treats Th17 cells by regulating their polarization.

5. The use according to claim 4, characterized in that, The reagent that regulates HIF-1α expression levels reduces Th17 cell polarization by decreasing the expression of the HIF-1α gene.

6. The use according to claim 3, characterized in that, Treatment of gout includes improving at least one of the following gout-related indicators: Lower uric acid levels; Reduce inflammatory cell infiltration in the footpads; Relieve swelling; Increase pain threshold; Reduce serum inflammatory infiltration; and Reduce IL-17A expression.

7. The use according to claim 3, characterized in that, Treatment of gout includes improving gout-related indicators in subjects, wherein the subjects are mammals, preferably mice and / or humans.

8. The use according to claim 3, characterized in that, The reagents used to regulate HIF-1α levels include diosgenin and PX-478.

9. A kit comprising reagents for determining HIF-1α expression levels and / or reagents for regulating HIF-1α expression levels.

10. A gout prediction device, characterized in that, The device includes: A sample collection unit, the sample collection unit being adapted to collect biological samples from a subject; A determining unit is used to determine the expression level of the HIF-1α gene in the obtained biological sample; The prediction unit compares the HIF-1α expression level with a reference value.

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