Application of ACSL1 gene in preparation of gouty arthritis prediction and diagnosis reagent and treatment medicine

Abstract

The invention relates to the field of gouty arthritis diagnosis and treatment, in particular to application of an ACSL1 gene in preparation of gouty arthritis prediction and diagnosis reagents and treatment drugs. Experiments prove that the expression level of the ACSL1 gene or mRNA / protein thereof is positively correlated with inflammatory response and ferroptosis of gouty arthritis, and the ACSL1 gene or mRNA / protein thereof can be used as a biomarker for disease risk prediction, activity evaluation or diagnosis. By inhibiting the expression of ACSL1 or blocking the activity of ACSL1, the secretion of inflammatory factors and the lipid peroxidation level can be remarkably reduced, and the pathological process is inhibited. The invention also relates to a diagnostic kit containing the ACSL1 detection reagent and a therapeutic pharmaceutical composition containing a targeted delivery system, and provides a new target and a solution for accurate diagnosis and treatment of gouty arthritis.

Description

Technical Field

[0001] The present invention relates to the field of diagnosis and treatment of gouty arthritis, and in particular to the use of the ACSL1 gene in preparing predictive and diagnostic reagents and therapeutic drugs for gouty arthritis. Background Art

[0002] Gouty arthritis (GA) is a disease caused by impaired purine metabolism and / or uric acid excretion. It primarily manifests as the accumulation of MSU in the joints, triggering acute joint inflammation. GA is the most typical clinical manifestation of gout, characterized by severe joint pain, swelling, fever, and redness. GA attacks not only cause immense pain for patients but can also lead to joint damage and even disability, severely impacting daily life and work. Research on GA in China has primarily focused on its etiology, epidemiology, and clinical treatment. Epidemiological data in my country show that the prevalence of gout ranges from 0.86% to 2.20%, with rates ranging from 0.83% to 1.98% in men and 0.07% to 0.72% in women, for a male-to-female ratio of 20:1. With the increasing prevalence of gout, research in China has increasingly focused on the immune mechanisms and treatment strategies underlying GA. Studies have shown that MSU crystals deposited in the joint cavity can activate the NLRP3 inflammasome, inducing the release of inflammatory cytokines such as IL-1β, triggering a strong local immune response and joint inflammation.

[0003] Currently, clinical treatments for GA primarily rely on colchicine and nonsteroidal anti-inflammatory drugs (NSAIDs). While these drugs offer significant therapeutic effects, long-term use can cause side effects such as gastrointestinal reactions and renal failure, and they are ineffective in preventing or reversing disease progression. Therefore, identifying novel biomarkers and therapeutic targets, and exploring strategies for early diagnosis and prevention of GA, are of great clinical value. Summary of the Invention

[0004] The present invention aims to provide the use of the ACSL1 gene in the preparation of predictive and diagnostic reagents and therapeutic drugs for gouty arthritis, aiming to provide a new solution to the above clinical needs.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions: In a first aspect, the present invention aims to provide an application of the ACSL1 gene or its expression product as a marker or therapeutic target for gouty arthritis.

[0006] ACSL1 refers to Acyl-CoA synthetase long-chain family member 1 (ACSL1), which is one of the key enzymes in fatty acid metabolism. It is mainly responsible for converting free long-chain fatty acids into acyl-CoA esters and participates in lipid biosynthesis and fatty acid degradation.

[0007] Furthermore, the application is: the CSL1 gene or its expression product is used to prepare any of the following products: Gouty arthritis risk prediction drugs or assessment reagents; Diagnostic reagents for gouty arthritis; Therapeutic drugs that inhibit inflammatory response or ferroptosis in gouty arthritis.

[0008] Furthermore, the prediction reagent, evaluation reagent or diagnostic reagent is used to detect the mRNA expression level of the ACSL1 gene or the expression level of the ACSL1 protein in a sample of a subject.

[0009] Furthermore, the kit comprises: qPCR primer pair for detecting ACSL1 mRNA; Alternatively, a specific antibody for detecting ACSL1 protein.

[0010] Furthermore, the therapeutic drug contains an active substance that inhibits ACSL1 expression as an active ingredient.

[0011] Furthermore, the inhibition includes at least any one of the following: Reduce ACSL1 mRNA transcription levels; or interfere with the mRNA translation process; or blocking the enzymatic activity of ACSL1 protein.

[0012] Preferably, the therapeutic drug is siRNA, shRNA or small molecule inhibitor targeting ACSL1.

[0013] Preferably, the therapeutic drug is used to inhibit macrophage ferroptosis and / or alleviate the inflammatory response of gouty arthritis.

[0014] Preferably, the therapeutic drug further comprises a pharmaceutically acceptable carrier or delivery system, for example, the carrier or delivery system includes but is not limited to liposomes (such as Lipo8000), nanoparticles, viral vectors (such as lentivirus, adeno-associated virus) or extracellular vesicles (EVs).

[0015] In a second aspect, the present invention further provides a method for screening a drug for treating gouty arthritis, comprising the following steps: S1: THP-1-derived macrophages or mouse bone marrow-derived macrophages were stimulated with sodium urate crystals; S2: applying the drug to be screened to the stimulated cells; S3: Detect the expression level of ACSL1 in cells, and screen potential drugs that can inhibit ACSL1 expression based on the detection results.

[0016] The principle and beneficial effects of this technical solution are: This invention reveals for the first time that the ACSL1 gene and its expression products are key regulatory factors in gouty arthritis, filling the gap in biomarkers and therapeutic targets in this field and providing a new direction for disease mechanism research and clinical intervention. The detection method based on ACSL1 expression level can accurately assess the risk and activity of the disease by quantifying the mRNA or protein levels in peripheral blood, synovial fluid or joint tissue, providing an objective basis for early diagnosis and intervention, and significantly improving the accuracy of clinical diagnosis and treatment. At the therapeutic level, by inhibiting ACSL1 expression (such as siRNA) or blocking its activity (such as small molecule inhibitors), ) strategy, verified by human / mouse dual model experiments, can effectively reduce the secretion of inflammatory factors such as IL-1β, reduce the accumulation of lipid peroxides (MDA), and increase the level of glutathione (GSH), thereby significantly inhibiting the inflammatory response and ferroptosis process, with clear efficacy and mechanism of action; in addition, macrophage-targeted delivery systems (such as modified liposomes and viral vectors) significantly enhance the enrichment efficiency of drugs in inflamed joints by coupling specific ligands on the surface, reduce off-target toxicity, further improve the safety and effectiveness of treatment, and provide a better solution for the clinical application of gouty arthritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Data processing of the GSE160170 dataset and functional enrichment analysis of DEGs, including: (A) volcano plot (left) and heat map (right) of the GSE160170 dataset; (B) GO enrichment analysis (left) and KEGG enrichment analysis (right) of DEGs.

[0018] Figure 2 Screening of Hub genes and their expression in the validation set, including: (A) Venn diagram of ferroptosis-related genes in GA; (B) Machine learning screening of Hub genes in GA; (C) Box plot of the expression levels of Hub genes in GA.

[0019] Figure 3 The figure shows the relationship between the proportion of immune cells and the expression of Hub genes in GA patients.

[0020] Figure 4Figure 2. ACSL1 expression in PBMCs from patients with GA. (A, B) RT-qPCR detection of DUSP1 and ACSL1 mRNA expression levels in PBMCs from patients with GA; (C) Western blot detection of ACSL1 protein expression in PBMCs from patients with GA; (D) Semi-quantitative analysis of the grayscale values ​​of the protein bands in Figure C. Compared with the control group, ns, no statistical difference; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

[0021] Figure 5 The expression of ACSL1 and inflammatory factors in GA mice, including: (A) Ankle joint swelling of mice 24 hours after MSU modeling; (B) Ankle joint swelling index of GA mice; (C) RT-qPCR detection of ACSL1 mRNA level in synovial tissue of MSU group; (D) Western blot detection of ACSL1 protein expression in synovial tissue of MSU group; (E) Semi-quantitative analysis of grayscale value of protein bands in Figure C; (F) IF double staining of CD68 and ACSL1 to detect ACSL1 level in macrophages of joint tissue of GA mice and control mice; (G) ELISA detection of TNF-α, IL-1β and IL-6 levels in serum of GA mice; (H) RT-qPCR detection of ACSL1 mRNA level in peripheral blood PBMCs of GA mice; (I) Western blot detection of ACSL1 protein expression in peripheral blood PBMCs of GA mice; (J) Semi-quantitative analysis of grayscale value of protein bands in Figure I. Compared with the control group, ns, no statistical difference; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

[0022] Figure 6 MSU upregulates ACSL1 expression and promotes ferroptosis in THP-1 cells ( ±s, n = 3), among which, (A) CCK8 assay for the effect of MSU on THP-1 cell viability; (B) ELISA assay for the level of inflammatory factor IL-1β in the cell supernatant after MSU stimulation of THP-1 cells; (C) RT-qPCR assay for the mRNA level of ACSL1; (D) RT-qPCR assay for the mRNA level of ferroptosis-related genes (GPX4, Nrf2, ACSL4); (E) Western blot assay for the protein expression of ACSL1 and ferroptosis-related genes (GPX4, Nrf2); (F, G) Semi-quantitative analysis of the gray value of the protein bands in Figure E; (HK) kit assay for the effects of MSU treatment on MDA, Fe 2Changes in GSH, GSSG, and GSH / GSSG ratio compared with the control group (ns, no statistical difference); *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

[0023] Figure 7 MSU upregulates the expression of ACSL1 and promotes ferroptosis in BMDMs cells ( ±s, n = 3) (A) CCK8 assay to detect the effect of MSU on BMDMs cell viability; (B) ELISA assay to detect the level of inflammatory factor IL-1β in the cell supernatant after MSU stimulation of BMDMs; (C) RT-qPCR assay to detect the mRNA level of ACSL1; (D) RT-qPCR assay to detect the mRNA level of ferroptosis-related genes (GPX4, Nrf2, ACSL4); (E) Western blot assay to detect the protein expression of ACSL1 and ferroptosis-related genes (GPX4, Nrf2); (F, G) Semi-quantitative analysis of the gray value of the protein bands in Figure E; (HK) kit to detect the effects of MSU treatment on MDA, Fe 2 Changes in GSH, GSSG, and GSH / GSSG ratio compared with the control group (ns, no statistical difference); *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

[0024] Figure 8 To interfere with the ACSL1 gene to inhibit ferroptosis in THP-1 cells ( ±s, n = 3), where: (A) RT-qPCR detection of the interference efficiency of ACSL1 interference fragments in THP-1 cells; (B) RT-qPCR detection of the mRNA levels of ferroptosis-related genes (GPX4, Nrf2) in THP-1 cells after ACSL1 interference; (C) Western blot detection of the protein expression of ACSL1 and ferroptosis-related genes (GPX4, Nrf2) in THP-1 cells after ACSL1 interference; (D, E) Semi-quantitative analysis of the gray value of the protein band in Figure C; (FI) Kit detection of MDA, Fe 2 Changes in GSH, GSSG, and GSH / GSSG ratio compared with the control group (ns, no statistical difference); *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

[0025] Figure 9 To interfere with ACSL1 gene to inhibit ferroptosis in BMDMs cells ( ±s, n = 3), where: (A) RT-qPCR detection of the interference efficiency of BMDMs transfected with ACSL1 interference fragments; (B) RT-qPCR detection of the mRNA levels of ferroptosis-related genes (GPX4, Nrf2) in BMDMs after ACSL1 interference; (C) Western blot detection of the protein expression of ACSL1 and ferroptosis-related genes (GPX4, Nrf2) in BMDMs after ACSL1 interference; (D, E) Semi-quantitative analysis of the gray value of the protein band in Figure C; (FI) Kit detection of MDA, Fe 2 + concentration, GSH, GSSG, and GSH / GSSG ratio. Compared with the control group, ns, no statistical difference; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

[0026] Figure 10 To partially reverse MSU-induced ferroptosis in THP-1 cells by interfering with ACSL1 ( ±s, n = 3), where: (A) ELISA was used to detect the level of IL-1β in the cell supernatant after MSU stimulation of THP-1 cells combined with ACSL1 interference; (B) RT-qPCR was used to detect the mRNA level of GPX4; (C) Western blot was used to detect the protein expression of GPX4; (D) Semi-quantitative analysis of the gray value of the protein band in Figure C; (EH) Kits were used to detect the expression of MDA and Fe in cells 2 + concentration, GSH, GSSG, and GSH / GSSG ratio. Compared with the control group, ns, no statistical difference; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

[0027] Figure 11 To interfere with ACSL1 and partially reverse MSU-induced ferroptosis in BMDMs ( ±s, n = 3), where: (A) ELISA was used to detect the level of IL-1β in the cell supernatant after MSU-stimulated BMDMs combined with ACSL1 interference; (B) RT-qPCR was used to detect the mRNA level of GPX4; (C) Western blot was used to detect the protein expression of GPX4; (D) Semi-quantitative analysis of the gray value of the protein band in Figure C; (EH) Kits were used to detect the expression of MDA and Fe in cells 2+ concentration, GSH, GSSG, and GSH / GSSG ratio. Compared with the control group, ns, no statistical difference; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

[0028] Figure 12 To overexpress ACSL1 gene to promote ferroptosis in THP-1 cells ( ±s, n = 3), where: (A, B) RT-qPCR detection of the mRNA levels of ACSL1 and ferroptosis-related genes (GPX4, Nrf2) in THP-1 cells transfected with ACSL1 overexpression virus; (C) Western blot detection of the protein expression of ACSL1 and ferroptosis-related genes in THP-1 cells transfected with ACSL1 overexpression virus; (D, E) Semi-quantitative analysis of the gray value of the protein band in Figure C; (FI) kit detection of MDA and Fe 2 Changes in GSH, GSSG, and GSH / GSSG ratio. Compared with the control group, ns, no statistical difference; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

[0029] Figure 13 To overexpress ACSL1 gene to promote ferroptosis in BMDMs cells ( ±s, n = 3), where: (A, B) RT-qPCR detection of mRNA levels of ACSL1 and ferroptosis-related genes (GPX4, Nrf2) in BMDMs transfected with ACSL1 overexpression virus; (C) Western blot detection of protein expression of ACSL1 and ferroptosis-related genes in THP-1 cells transfected with ACSL1 overexpression virus; (D, E) Semi-quantitative analysis of grayscale values ​​of protein bands in Figure C; (FI) kit detection of MDA and Fe 2 Changes in GSH, GSSG, and GSH / GSSG ratio. Compared with the control group, ns, no statistical difference; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. DETAILED DESCRIPTION

[0030] The following examples are only used to more clearly illustrate the technical solutions of the present invention and are therefore only examples and are not intended to limit the scope of protection of the present invention. The specific embodiments listed in the present invention are only examples of the present invention, and the present invention is not limited to the specific embodiments described below. For those skilled in the art, any equivalent modifications and substitutions made to the embodiments described below are also within the scope of the present invention. Therefore, equivalent transformations and modifications made without departing from the spirit and scope of the present invention should be included within the scope of the present invention. In order to better illustrate the present invention, numerous specific details are given in the specific embodiments below. Those skilled in the art should understand that the present invention can also be implemented without certain specific details. In other embodiments, methods, means, equipment and steps well known to those skilled in the art are not described in detail in order to highlight the main purpose of the present invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art.

[0031] Unless otherwise specified, the units used in this specification are all international standard units, and the numerical values ​​and numerical ranges appearing in the present invention should be understood to include the inevitable systematic errors in industrial production. Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials, reagents, or instruments used, if the manufacturer is not indicated, are all reagents and materials that can be obtained from commercial channels. If specific conditions are not specified in the examples, they are all carried out according to conventional conditions or conditions recommended by the manufacturer. At the same time, the present invention does not limit the sources of the raw materials used. Unless otherwise specified, the raw materials used in the present invention are all common commercial products in the field of technology.

[0032] It is important to note that: (1) The statistical methods used in the following examples are: Statistical analyses were performed using R software (version 4.2.3; https: / / www.r-project.org / ), SPSS 27.0, and GraphPad Prism 8.0.2. When measurement data conformed to a normal distribution, t-tests were used for comparisons between the two groups; when data did not conform to a normal distribution, Welch's t-tests were used for comparisons between the two groups. Correlations were analyzed using Spearman correlation analysis. P < 0.05, P < 0.01, P < 0.001, and P < 0.0001 indicated statistically significant differences.

[0033] (2) The primers used in the following examples were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd., and their specific sequences are shown in Tables 1 and 2. Based on the gene sequence, the siRNA sequences were provided by Suzhou Haixing Biotechnology Co., Ltd., and their specific sequences are shown in Tables 3 and 4.

[0034] Table 1 Primer sequences of human related genes

[0035] Table 2 Primer sequences of mouse-related genes

[0036] Table 3 Sequences of human siRNA

[0037] Table 4 Sequences of mouse siRNA

[0038] Example 1 1.1 Microarray data preprocessing and functional enrichment analysis of DEGs In this study, the GA microarray dataset GSE160170 was downloaded from the GEO database (http: / / www.ncbi.nlm.nih.gov / geo / ). The downloaded microarray data GSE160170 were normalized using R software to obtain standardized data. The GSE160170 dataset was analyzed using the Limma package. Using |logFC| > 0.585 and adjusted P < 0.05 as the criteria, 277 DEGs were screened, including 206 up-regulated genes and 71 down-regulated genes. The experimental results are shown in Figure 1 A.

[0039] GO analysis of differentially expressed genes was performed using R language, and KEGG enrichment analysis was performed using the org.Hs.eg.db database file on the bioconductor platform and the David (https: / / david.ncifcrf.gov / ) online tool. Biological processes (BP) mainly focused on transcriptional regulation of RNA polymerase II promoter, positive transcriptional regulation of RNA polymerase II promoter, signal transduction, negative transcriptional regulation of RNA polymerase II promoter, and DNA template. Cellular components (CC) mainly focused on nucleus, cytoplasm, cytoplasm, nucleoplasm, and Golgi apparatus. Molecular functions (MF) focused on protein binding, metal ion binding, identical protein binding, sequence-specific DNA binding of the proximal region of the RNA polymerase core promoter, and DNA binding. In addition, KEGG pathway analysis showed that DEGs were closely related to osteoclast differentiation, FoxO signaling pathway, and interaction with non-alcoholic fatty liver disease. The experimental results are shown in

[15] . Figure 1 B.

[0040] 1.2 Screening of Hub genes and their expression in the validation set First, the DEGs of GSE160170 were analyzed using the “Limma” package and presented in volcano plots and heat maps. Then, a gene set containing 291 genes was obtained from the ferroptosis database and intersected with the DEGs of GSE160170 to identify Ferr-DEGs. A total of 8 Ferr-DEGs were found and represented by Venn diagrams. The experimental results are shown in Figure 2 A.

[0041] The LASSO regression algorithm identified two ferroptosis-related genes, while the SVM-RFE algorithm identified six ferroptosis-related genes. Both algorithms identified two Hub genes: DUSP1 and ACSL1. Figure 2 B.

[0042] Finally, the expression of Hub genes in the GSE160170 dataset was verified, and it was found that the expression levels of DUSP1 and ACSL1 in GA patients were higher than those in healthy subjects (P < 0.01). The experimental results are shown in Figure 2 C.

[0043] 1.3 Analysis of the proportion of immune cells in GA patients and their correlation with Hub gene expression The onset of GA can lead to changes in the proportions and functions of a range of immune cells. To identify biomarkers whose expression correlates with immune cell proportions, we first used the CIBERSORT algorithm to analyze the changes in the proportions of 22 immune cells in six GA patients and six healthy controls. The results showed a significant increase in the proportion of monocytes in GA patients compared with controls (P < 0.05), while other markers, such as immature B lymphocytes, showed no statistically significant differences between the two groups (P > 0.05).

[0044] In addition, we further studied the relationship between the proportion of immune cells and Hub gene expression in GA patients. Figure 3 The experimental results showed that the expression of DUSP1 and ACSL1 was significantly positively correlated with the proportion of monocytes (r = 0.65, 0.776, both P < 0.05).

[0045] 1.4 Analysis of ACSL1 expression levels in PBMCs of GA patients and its correlation with inflammatory and metabolic markers Blood samples, clinical data, and examination parameters were collected from 80 male patients with GA who visited Taizhou People's Hospital Affiliated to Nanjing Medical University between March and December 2023. All GA patients met the 2015 ACR / EMLAR diagnostic criteria for gout. Exclusion criteria included: 1. Patients with Cushing's syndrome and previous use of glucocorticoids and nonsteroidal anti-inflammatory drugs; 2. Severe cardiac, hepatic, renal, or cerebrovascular disease; 3. Rheumatoid arthritis, osteoarthritis, or traumatic arthritis; 4. Pregnant or lactating women; 5. Severe psychiatric illness. During the same period, 60 healthy male controls without chronic diseases such as hyperuricemia and metabolic syndrome were recruited. All subjects provided written informed consent. This study was approved by the Ethics Committee of Taizhou People's Hospital Affiliated to Nanjing Medical University (KY2023-149-01) and conducted in accordance with the ethical principles of the 1975 Declaration of Helsinki.

[0046] RT-PCR was used to detect the mRNA expression of related genes. For the experimental results, please refer to Figure 4 : In the peripheral blood PBMCs of 80 GA patients and 60 healthy controls, the relative expression levels of DUSP1 were 0.6818±0.2088 and 0.5710±0.1866, respectively, and the relative expression levels of ACSL1 were 1.472±0.8347 and 0.9435±0.3443, respectively. Compared with the healthy control group, the expression level of DUSP1 in PBMCs of GA patients was significantly increased, and the difference was statistically significant (t = 3.249, P < 0.01, Figure 4 A); The expression level of ACSL1 was significantly upregulated, and the difference was statistically significant (t = 5.117, P < 0.0001, Figure 4 B). Therefore, the focus of subsequent studies was on the ACSL1 gene.

[0047] Western blot further confirmed that the expression level of ACSL1 protein was also significantly increased. Compared with the healthy control group, the level of ACSL1 protein in PBMCs of GA patients was increased, and the difference was statistically significant (P < 0.05, Figure 4 C, D).

[0048] The UA, blood urea (Urea), blood glucose (GLU) levels, as well as inflammation and lipid metabolism indicators of all subjects were collected and evaluated, and Spearman correlation analysis was performed. The experimental results are shown in Table 5: Table 5 Correlation analysis between ACSL1 and clinical test indicators

[0049] The experimental results showed that ACSL1 levels had no significant correlation with age, WBC, LY, Mo, GLOB, Urea, GLU, TG and apoB100 (all P>0.05); but were positively correlated with UA, TNF-α, IL-1β, IL-6, NEUT and Crea (all P<0.05), and negatively correlated with ALB, TC, HDL-C, LDL-C, apoA1 and eGFR (all P<0.05).

[0050] The above experimental results showed that the expression level of ACSL1 in PBMCs of GA patients was significantly upregulated and closely correlated with inflammatory and metabolic indicators, suggesting that ACSL1 may participate in the occurrence of GA by regulating inflammatory response and metabolic disorders.

[0051] Example 2 2.1 Animal experiments to investigate the role of ACSL1 in GA To investigate the role of ACSL1 in GA, this study established a GA mouse model by injecting MSU crystals into the ankle joint cavity. Mice were housed in an animal room with a 12-h light-dark cycle, an ambient temperature of (22 ± 2)°C, good ventilation, and a relative humidity of 40–60%. All cages, feed, and drinking water were sterilized. After purchase, mice were acclimated to feeding for one week before experiments were conducted. All animal experiments were approved by the Experimental Animal Committee of Jiangsu Hanjiang Biotechnology Co., Ltd. (HJSW-25022001).

[0052] After one week of adaptive feeding, mice were randomly divided into a normal group (Control group) and a model group (MSU group), with six mice in each group. Gas anesthesia was administered using an anesthesia system. Mice developed signs of general anesthesia after 5 seconds of inhalation of 3% isoflurane. After local disinfection, 40 µL of a suspension (containing 25 mg / mL MSU) was drawn up using a 1 mL disposable sterile syringe and injected into the medial aspect of the right ankle joint. A bulge on the contralateral side indicated successful injection, thus establishing a GA inflammation model. Mice in the NC group were injected with an equal volume of normal saline in the same manner.

[0053] 24 hours after modeling, mice were anesthetized by inhalation of isoflurane. Eye blood was removed and centrifuged at 3000 rpm for 10 minutes to collect serum for later use. Next, approximately 1 cm long sections were cut from the ankle joint of the mouse's hind limb, carefully removing the fur and muscle attached to the ankle joint. The ankle joint cavity was opened with scissors and transferred to a 1.5 mL enzyme-free EP tube. Stored at -80°C until further use, the following assays were performed: After modeling, the ankle joint swelling of mice in the MSU injection group was significantly increased compared with that in the control group (P < 0.0001), indicating that the GA mouse model was successfully established ( Figure 5 A, B).

[0054] RT-qPCR and Western blot results showed that the expression level of ACSL1 in the synovial tissue of the GA mice group was increased, and the difference was statistically significant ( Figure 5 CE).

[0055] IF double staining results further confirmed that ACSL1 expression was significantly upregulated in macrophages in the joint tissues of GA mice ( Figure 5 F). The levels of inflammatory factors TNF-α, IL-1β, and IL-6 in human and mouse serum were detected by ELISA. The results of inflammatory factor detection showed that the expression levels of TNF-α, IL-1β, and IL-6 in the serum of GA mice were significantly upregulated ( Figure 5 G). In addition, the expression level of ACSL1 in PBMCs of GA mice was significantly higher than that of the Control group ( Figure 5 HJ).

[0056] The above results showed that ACSL1 was significantly overexpressed in the MSU-induced GA mouse model and was closely related to the release of proinflammatory factors, suggesting that ACSL1 may participate in the occurrence and development of GA by regulating the inflammatory response.

[0057] 2.2 MSU stimulation upregulates ACSL1 expression and promotes macrophage ferroptosis To further confirm that ACSL1 is involved in the occurrence and development of GA, THP-1 cells (human monocytic leukemia cells) and BMDMs were stimulated with MSU crystal suspension to successfully construct a cellular inflammation model.

[0058] The experimental results are as follows Figure 6 and Figure 7 As shown: The CCK8 results suggested that 200 ug / mL was the appropriate concentration for subsequent cell modeling ( Figure 6 A, Figure 7 A).

[0059] ELISA results showed that after MSU stimulation, the IL-1β level in the cell supernatant of THP-1 cells and BMDMs increased ( Figure 6 B, Figure 7 B), indicating that the inflammation model was successfully constructed.

[0060] In the study of molecular mechanisms, the relative expression of ACSL1 mRNA was detected by real-time fluorescence quantitative PCR (qPCR) using the following primers: Human ACSL1 upstream primer: 5'-CTGGTGGTGCTGGTGATG-3', Human ACSL1 downstream primer: 5'-GGTGCTGGTGGTGATGGT-3'; RT-qPCR and Western blot results showed that MSU stimulation significantly upregulated the expression levels of ACSL1 mRNA and protein in THP-1 cells and BMDMs ( Figure 6 CF, Figure 7 CF), while the mRNA expression of ferroptosis-related genes (GPX4, Nrf2) decreased, while the mRNA expression of ACSL4 was upregulated ( Figure 6 D, Figure 7 D). Western blot results further confirmed that GPX4 and Nrf2 protein levels decreased, among which Nrf2 protein showed no statistically significant change in BMDMs ( Figure 6 G, Figure 7 G).

[0061] Based on the above results, we speculated that ACSL1 may play an important role in MSU-induced macrophage damage. To verify our hypothesis and further explore whether ACSL1 is involved in the ferroptosis process, we measured the expression of MDA, Fe 2 + Concentration, GSH, GSSG and GSH / GSSG ratio.

[0062] The experimental results can be found in Figure 6 and Figure 7 The results showed that oxidative stress was aggravated in THP-1 cells and BMDMs treated with MSU, and the intracellular MDA and Fe 2 + Concentration increased significantly ( Figure 6 H, I, Figure 7 H, I), GSH content decreased significantly, while GSSG content increased significantly ( Figure 6 J, Figure 7 J), GSH / GSSG ratio decreased significantly ( Figure 6 K, Figure 7 K).

[0063] The above results confirmed that MSU promoted macrophage ferroptosis by upregulating the expression of ACSL1, suggesting that ACSL1 may play a key role in the pathogenesis of GA by mediating the ferroptosis mechanism.

[0064] Example 3 To investigate the role of ACSL1 in macrophage ferroptosis, siRNA technology was used to interfere with its expression. The experimental methods and results are briefly described as follows: 3.1 Gene knockdown Targeting human monocytic cell line (THP-1) and mouse bone marrow-derived macrophages (BMDMs), the ACSL1 gene was knocked down by siRNA transfection to verify its regulatory effect on MSU-induced inflammatory response and ferroptosis.

[0065] (1) Cell preparation and grouping: THP-1 cells (human): 1×10 6 Inoculate 1000 cells / well in a 6-well plate and set up 3 groups (3 replicates per group): ① Screening group: si-NC (negative control), si-hACSL1-334, si-hACSL1-39, si-hACSL1-11 (three siRNAs targeting human ACSL1); ② Validation group: si-NC, si-hACSL1-11 (screened high-efficiency interference sequences); ③ Functional groups: si-NC+PBS (no stimulation control), si-NC+MSU (normal expression+MSU stimulation), si-hACSL1-11+MSU (knockdown+MSU stimulation).

[0066] BMDMs cells (mouse): 8×10 5 Cells were seeded into 6-well plates (the density was slightly lower due to the weak proliferation ability of primary cells) and grouped in the same way as THP-1, except that siRNA was replaced by mouse si-mACSL1-13 (a high-efficiency sequence screened in preliminary experiments).

[0067] (2) siRNA transfection operation: THP-1 transfection: Use Lipo8000 liposomes (suitable for immortalized cells with high transfection efficiency): Tube A (250 μL serum-free 1640 medium + 4 μL Lipo8000) was mixed with tube B (250 μL serum-free 1640 medium + 1 μL siRNA), allowed to stand at room temperature for 20 min, and then added dropwise to the cells. After incubation at 37°C, 5% CO2 for 6 h, the medium was replaced and cultured for another 48 h.

[0068] BMDMs transfection: Because primary cells are sensitive to transfection reagents, electroporation is used (to reduce toxicity): The cells were centrifuged and resuspended in electroporation buffer (100 μL / tube), 1 μL of siRNA was added, and the parameters were set (voltage 200 V, pulse time 20 ms). Preheated culture medium was added immediately after electroporation, and the culture medium was changed after culturing at 37°C for 24 h and then continued to be cultured for 48 h.

[0069] (3) Detection method: Molecular level: RNA (Trizol method) and protein (RIPA lysis buffer) were extracted 48 hours after transfection, and knockdown efficiency was detected by qPCR (primers specifically designed for human / mouse ACSL1) and Western blotting; Functional level: After MSU stimulation (200 μg / mL, 24 h), the IL-1β level in the supernatant was detected by ELISA, and the intracellular MDA (lipid peroxidation product) and GSH (antioxidant) levels were detected by colorimetry.

[0070] (4) Experimental results: The experimental results can be found in Figure 8 and Figure 9 , qRT-PCR results showed that compared with si-NC, si-hACSL1-11 and si-mACSL1-13 significantly reduced the mRNA expression level of ACSL1 in THP-1 cells and BMDMs. Western blot results also showed that the expression of ACSL1 protein was significantly decreased, indicating that the interference model was successfully established ( Figure 8 A, C, D; Figure 9 A, C, D). After ACSL1 interference, GPX4 mRNA and protein expressions were upregulated, while Nrf2 expression did not change significantly ( Figure 8 B, C, E; Figure 9 B, C, E), while MDA, Fe 2 + content decreased, GSH increased, GSSG decreased, and the GSH / GSSG ratio increased ( Figure 8 FI; Figure 9 FI), suggesting that ACSL1 interference can inhibit ferroptosis in macrophages.

[0071] The experimental results can be found in Figure 10 and Figure 11 , ELISA results showed that compared with the si-NC+PBS group, the IL-1β level in the cell supernatant was significantly increased in the si-NC+MSU group, while the IL-1β level in the MSU combined with ACSL1 interference group was significantly decreased ( Figure 10 A. Figure 11 A). At the same time, the expression level of GPX4 decreased after MSU stimulation, but increased significantly after ACSL1 interference ( Figure 10 BD, Figure 11 BD). Ferroptosis-related indicators showed that MDA, Fe 2 + increases, GSH decreases, GSSG increases, and the GSH / GSSG ratio decreases ( Figure 10 EH, Figure 11These changes were significantly reversed after combined ACSL1 knockdown, indicating that ACSL1 plays a key regulatory role in MSU-induced macrophage ferroptosis. These results suggest that MSU promotes macrophage ferroptosis by upregulating ACSL1 expression, thereby exacerbating the inflammatory response, while ACSL1 knockdown can effectively alleviate this process. This suggests that ACSL1 plays a key regulatory role in the pathogenesis of GA and may provide a new target for the diagnosis and treatment of GA.

[0072] 3.3 Viral infection for gene overexpression To further investigate the role and mechanism of ACSL1 in macrophage ferroptosis, adeno-associated virus (AAV) technology was used to mediate ACSL1 overexpression, which was then verified by knockdown experiments.

[0073] (1) Lentiviral vector construction and packaging: Lentiviral vectors pLV-hACSL1 and pLV-mACSL1 (driven by the CMV promoter and carrying the puromycin resistance gene) containing the full-length cDNAs of human ACSL1 (hACSL1) and mouse ACSL1 (mACSL1) were constructed; 293T cells (packaging cells) were cultured at 5 × 10 5 The cells were seeded into 6-well plates and co-transfected with pLV-ACSL1, psPAX2 (packaging plasmid) and pMD2.G (envelope plasmid) using Lipo8000. The viral supernatant was collected 48 h later (filtered with a 0.45 μm membrane and stored at -80°C).

[0074] (2) Cell infection and screening: THP-1 infection: cells were infected at 1×10 6 The cells were inoculated at 100 μg / well, and viral supernatant (MOI=15) and Polybrene (8 μg / mL) were added. The culture medium was changed after 48 h of infection at 37°C, and the cells were screened with puromycin (3 μg / mL) for 7 days to obtain THP-1 cells stably overexpressing hACSL1 (THP-1-hACSL1). The uninfected group was designated as THP-1-NC (negative control).

[0075] BMDMs infection: Primary cells were cultured at 8 × 10 5 The cells were inoculated into 100 cells / well. Because BMDMs are less sensitive to viral infection, a higher MOI (MOI=20) was used. The medium was changed 48 hours after infection, and puromycin (2 ug / mL) was added for selection for 5 days to obtain BMDMs stably overexpressing mACSL1 (BMDMs-mACSL1). The uninfected group was designated as BMDMs-NC.

[0076] (3) Functional verification experiment: The two groups of cells were stimulated with MSU suspension (200 μg / mL) for 24 h, and the cells and supernatant were collected; Detection methods: qPCR (verification of overexpression efficiency), Western Blot (protein level), ELISA (IL-1β), colorimetric assay (MDA, GSH).

[0077] (4) The experimental results are as follows: The experimental results can be found in Figure 12 and Figure 13 , qRT-PCR results showed that compared with ACSL1-NC, overexpression of ACSL1 virus significantly upregulated the mRNA expression of ACSL1 in THP-1 cells and BMDMs, and downregulated the mRNA expression of GPX4 and Nrf2 ( Figure 12 A, B, Figure 13 A, B). Western blot further confirmed that ACSL1 overexpression could reduce GPX4 protein expression, but had no significant effect on Nrf2 protein levels ( Figure 12 CE, Figure 13 CE).

[0078] In addition, the results of ferroptosis-related index detection showed that overexpression of ACSL1 significantly increased intracellular MDA and Fe 2 + content, reducing GSH content, increasing GSSG level, and leading to a decrease in the GSH / GSSG ratio ( Figure 12 FI, Figure 13 FI). These results suggest that ACSL1 may promote macrophage ferroptosis by inhibiting the expression of GPX4 and enhancing ferroptosis-related molecular changes.

[0079] The above is only an embodiment of the present invention, and the common knowledge such as the specific technical solutions and / or characteristics in the solution are not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several variations and improvements can be made, which should also be regarded as the scope of protection of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the description can be used to interpret the content of the claims.

Claims

1. Use of an ACSL1 gene or its expression product as a marker or therapeutic target for gouty arthritis.

2. The use according to claim 1, characterized in that The CSL1 gene or its expression product is used to prepare any of the following products: Gouty arthritis risk prediction drugs or assessment reagents; Diagnostic reagents for gouty arthritis; Therapeutic drugs that inhibit inflammatory response or ferroptosis in gouty arthritis.

3. The use according to claim 2, characterized in that: The predictive reagent, evaluation reagent or diagnostic reagent is used to detect the mRNA expression level of the ACSL1 gene or the expression level of the ACSL1 protein in a sample of a subject.

4. The use according to claim 2, wherein: The kit comprises: qPCR primer pair for detecting ACSL1 mRNA; Alternatively, a specific antibody for detecting ACSL1 protein.

5. The use according to claim 2, characterized in that: The therapeutic drug contains an active substance that inhibits ACSL1 expression as an active ingredient.

6. The drug according to claim 2, characterized in that: The inhibition includes at least any of the following: Reduce ACSL1 mRNA transcription levels; or interfere with the mRNA translation process; or blocking the enzymatic activity of ACSL1 protein.

7. The drug according to claim 2, wherein The therapeutic drug is siRNA, shRNA or small molecule inhibitor targeting ACSL1.

8. The use according to claim 2, characterized in that The therapeutic drug is used to inhibit macrophage ferroptosis and / or alleviate the inflammatory response of gouty arthritis.

9. The use according to claim 2, characterized in that The therapeutic drug also includes a pharmaceutically acceptable carrier or delivery system.

10. A method for screening drugs for treating gouty arthritis, characterized in that: The following steps are involved: S1: Use sodium urate crystals to stimulate THP-1-derived macrophages or mouse bone marrow-derived macrophages; S2: Apply the drug to be screened to the stimulated cells; S3: Detect the expression level of ACSL1 in cells, and screen potential drugs that can inhibit ACSL1 expression based on the detection results.

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